Pharmaceutical composition with cytotoxic activity on colorectal cancer cells

ABSTRACT

A compound of formula (I), 
                         
wherein R 1  is an optionally substituted aryl or an optionally substituted heteroaryl, R 2  is an optionally substituted aryl or an optionally substituted heteroaryl, and R 3  is an optionally substituted alkylamino, an optionally substituted cycloalkylamino, an optionally substituted arylamino, an optionally substituted heterocyclylamino, an optionally substituted heterocyclyl, and an optionally substituted dialkylamino. A pharmaceutical composition which includes the compound of formula (I) and a pharmaceutically acceptable carrier and/or excipient. A method of treating colorectal cancer in a subject, whereby a therapeutically effective amount of the compound of formula (I) is administered to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of Ser. No. 16/663,861,pending, having a filing date of Oct. 25, 2019.

STATEMENT OF ACKNOWLEDGEMENT

This project was funded by the Deanship of Scientific Research (DSR),King Abdulaziz University, Jeddah, the Kingdom of Saudi Arabia, undergrant number RG-7-166-38.

BACKGROUND OF THE INVENTION Technical Field

The present disclosure relates to a family of therapeutic compoundshaving a bis-propenamide scaffold, a pharmaceutical compositioncontaining the compounds, and a method for treating cancer with thecompounds.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Several Michael acceptors (MAs) have been developed or are currentlybeing developed as therapeutic agents. For instance, Afatinib (FIG. 1A)a target-specific MA selectively inhibits mutated HER2 kinase, and isapproved by the FDA for the treatment of non-small cell lung cancer. SeeMinkovsky, N.; Berezov, A., BIBW-2992, a dual receptor tyrosine kinaseinhibitor for the treatment of solid tumors. Curr Opin Investig Drugs2008, 9 (12), 1336-46; and Suzawa, K.; Toyooka, S.; Sakaguchi, M.;Morita, M.; Yamamoto, H.; Tomida, S.; Ohtsuka, T.; Watanabe, M.;Hashida, S.; Maki, Y.; Soh, J.; Asano, H.; Tsukuda, K.; Miyoshi, S.,Antitumor effect of afatinib, as a HER2-targeted therapy, in lungcancers harboring HER2 oncogene alterations. Cancer Sci 2015, eachincorporated herein by reference in their entirety. Other examplesinclude ibrutinib (BTK inhibitor) and neratinib (HER-2 inhibitor) thathave been developed for B-cell cancers and solid tumors, respectively.See Singh, J.; Petter, R. C.; Baillie, T. A.; Whiny, A., The resurgenceof covalent drugs. Nat Rev Drug Discov 2011, 10 (4), 307-17; andLanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.;Hat, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.;Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.;Whiteley, L. O.; Hayward, M. M.; Cravat, B. F., A road map to evaluatethe proteome-wide selectivity of covalent kinase inhibitors. Nat ChemBiol 2014, 10 (9), 760-7, each incorporated herein by reference in theirentirety. Mechanistically, the MAs cause cancer cell apoptosis byincreasing the oxidative stress inside these cells. The cellularprooxidant induction by MAs is attributed to ligation of SH groups ofcertain targets involved in regeneration of reduced glutathione (GSH).This results in accumulation of reactive oxygen species (ROS) whichcause cancer cell cycle exits and apoptosis. See Wondrak, G. T.,Redox-directed cancer therapeutics: molecular mechanisms andopportunities. Antioxid Redox Signal 2009, 11 (12), 3013-69,incorporated herein by reference in its entirety.

Acrolein (FIG. 1A) is a highly reactive and non-selective MA toxin andenvironmental pollutant with well-known carcinogenic activity.Interestingly, acrolein is also known to cause inhibition of theβ-subunit of the proliferative anti-apoptotic protein NFκB via covalentbinding with the nucleophilic Cys-61 and Arg-307 leading to cytotoxicityand cell death of cancer cells. See Chan, K.; Poon, R.; O'Brien, P. J.,Application of structure-activity relationships to investigate themolecular mechanisms of hepatocyte toxicity and electrophilic reactivityof alpha,beta-unsaturated aldehydes. J Appl Toxicol 2008, 28 (8),1027-39, incorporated herein by reference in its entirety. Thus despiteits apparent toxic effect, acrolein like other MAs has potential as atherapeutic agent. Replacement of the aldehyde group of acrolein with anamide group (acrylamide moiety) is known to attenuate the reactivity ofthe MA moiety leading to less toxic compounds. See Schwobel, J. A.;Wondrousch, D.; Koleva, Y. K.; Madden, J. C.; Cronin, M. T.; Schuurmann,G., Prediction of michael-type acceptor reactivity toward glutathione.Chem Res Toxicol 2010, 23 (10), 1576-85, incorporated herein byreference in its entirety. Safe natural MA products that are common indomestic condiments, such as trans-cinnamaldehyde (tCA) and curcumin(FIG. 1), are known to cause cancer cell death via redox mechanisms. SeeWondrak, G. T., Redox-directed cancer therapeutics: molecular mechanismsand opportunities, Antioxid Redox Signal 2009, 11 (12), 3013-69; Noh,J.; Kwon, B.; Han, E.; Park, M.; Yang, W.; Cho, W.; Yoo, W.; Khang, G.;Lee, D., Amplification of oxidative stress by a dual stimuli-responsivehybrid drug enhances cancer cell death. Nature communications 2015, 6,6907; Lin, L. T.; Tai, C. J.; Chang, S. P.; Chen, J. L.; Wu, S. J.; Lin,C. C., Cinnamaldehyde-induced apoptosis in human hepatoma PLC/PRF/5cells involves the mitochondrial death pathway and is sensitive toinhibition by cyclosporin A and z-VAD-fmk. Anticancer Agents Med Chem2013, 13 (10), 1565-74; Wondrak, G. T.; Villeneuve, N. F.; Lamore, S.D.; Bause, A. S.; Jiang, T.; Zhang, D. D., The cinnamon-derived dietaryfactor cinnamic aldehyde activates the Nrf2-dependent antioxidantresponse in human epithelial colon cells. Molecules 2010, 15 (5),3338-55; Kwon, H. K.; Hwang, J. S.; So, J. S.; Lee, C. G.; Sahoo, A.;Ryu, J. H.; Jeon, W. K.; Ko, B. S.; Im, C. R.; Lee, S. H.; Park, Z. Y.;Im, S. H., Cinnamon extract induces tumor cell death through inhibitionof NFkappaB and AP1. BMC Cancer 2010, 10, 392; Cabello, C. M.; Bair, W.B., 3rd; Lamore, S. D.; Ley, S.; Bause, A. S.; Azimian, S.; Wondrak, G.T., The cinnamon-derived Michael acceptor cinnamic aldehyde impairsmelanoma cell proliferation, invasiveness, and tumor growth. Free RadicBiol Med 2009, 46 (2), 220-31; Ka, H.; Park, H. J.; Jung, H. J.; Choi,J. W.; Cho, K. S.; Ha, J.; Lee, K. T., Cinnamaldehyde induces apoptosisby ROS-mediated mitochondrial permeability transition in humanpromyelocytic leukemia HL-60 cells. Cancer Lett 2003, 196 (2), 143-52;and Aggarwal, B. B.; Surh, Y.-J.; Shishodia, S., The molecular targetsand therapeutic uses of curcumin in health and disease. Springer Science& Business Media: 2007; Vol. 595, each incorporated herein by referencein their entirety. However, there still exists a need for new MAanticancer agents with a large therapeutic window, that is, that exhibitpotent anti-proliferative effects against cancerous cells withoutsignificantly effecting normal cells.

In view of the forgoing, one objective of the present disclosure is toprovide safe therapeutic compounds with antiproliferative activitiesbased on a bis-propenamide scaffold having a cinnamoyl-type appendage, apharmaceutical composition containing the compounds, and a method fortreating cancer with the compounds.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it is one object of the present invention to providecompounds with bis(propenamide) scaffolds based on trans-cinnamaldehyde(3-phenylpropenal or tCA) and curcumin structural features, which arenon-toxic and have excellent pharmacologic properties.

It is another object of the present invention to provide pharmaceuticalcompositions containing the compounds.

It is yet another object of the present invention to provide methods fortreating cancer with the compounds.

Thus, the present invention provides:

A compound of formula (I),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof,

wherein:

R¹ is an optionally substituted aryl or an optionally substitutedheteroaryl;

R² is an optionally substituted aryl or an optionally substitutedheteroaryl; and

R³ is an optionally substituted alkylamino, an optionally substitutedcycloalkylamino, an optionally substituted arylamino, an optionallysubstituted heterocyclylamino, an optionally substituted heterocyclyl,and an optionally substituted dialkylamino.

In some embodiments, R¹ is an optionally substituted phenyl or anoptionally substituted thienyl.

In some embodiments, R¹ is phenyl, a 4-halophenyl, a 4-alkoxyphenyl, a4-alkylphenyl, and a thienyl.

In some embodiments, R¹ is selected from the group consisting of phenyl,4-chlorophenyl, 4-methoxyphenyl, 4-methylphenyl, and 2-thienyl.

In some embodiments, R¹ is phenyl or 2-thienyl.

In some embodiments, R² is an optionally substituted phenyl, anoptionally substituted indolyl, or an optionally substituted pyridyl.

In some embodiments, R² is phenyl, a 4-halophenyl, a4-dialkylaminophenyl, 3-alkoxy-4-hydroxyphenyl, a 4-nitrophenyl, a1H-indolyl, and a pyridyl.

In some embodiments, R² is selected from the group consisting of phenyl,4-fluorophenyl, 4-dimethylaminophenyl, 3-methoxy-4-hydroxyphenyl,4-nitrophenyl, 1H-indol-3-yl, and 3-pyridyl.

In some embodiments, R² is phenyl or 1H-indol-3-yl.

In some embodiments, R³ is an optionally substituted C₁ to C₄alkylamino, an optionally substituted C₅ to C₁₀ cycloalkylamino, anoptionally substituted phenylamino, an optionally substitutedpyridylamino, an optionally substituted N-piperazinylamino, anoptionally substituted morpholinyl, an optionally substitutedpyrrolidinyl, and an optionally substituted di-(C₁ to C₃ alkyl)amino.

In some embodiments, R³ is selected from the group consisting ofcyclopentylamino, 1-adamantylamino, 3-pyridylamino,4-methyl-1-piperazinylamino, ethylamino, n-propylamino, n-butylamino,sec-butylamino, 3-cyanophenylamino, 4-fluorophenylamino, furfurylamino,2-morpholinoethylamino, 2-hydroxyethylamino, 4-morpholinyl,1-pyrrolidinyl, and methylethylamino.

In some embodiments, R³ is a C₃ to C₄ alkylamino, a C₅ to C₁₀cycloalkylamino, or a di-(C₁ to C₃ alkyl)amino

In some embodiments, R³ is n-propylamino.

The present invention also provides a pharmaceutical composition, whichincludes the compound of formula (I) and a pharmaceutically acceptablecarrier and/or excipient.

In some embodiments, the pharmaceutical composition is in the form of anaqueous suspension, and wherein the pharmaceutically acceptable carrierand/or excipient is a non-ionic surfactant in water.

In some embodiments, the pharmaceutical composition contains 5 to 15 wt.% of the compound of formula (I), relative to a total weight of thepharmaceutical composition.

The present invention also provides a method for treating colorectalcancer in a subject, by administering to the subject a therapeuticallyeffective amount of the compound of formula (I).

In some embodiments, the therapeutically effective amount of thecompound of formula (I) is from 0.1 to 2,000 mg/kg of the compound offormula (I) per body weight of the subject.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1A illustrates Michael acceptor (MA)-based drugs, flavors andpoisons;

FIG. 1B illustrates the bis-propenamide scaffold of the compounds of thepresent disclosure;

FIG. 1C illustrates a synthetic scheme used to for the synthesis of thecompounds of the present disclosure: (a) malonic acid, pyridine, heat;(b) oxalyl chloride, DCM, DMF (cat.) then glycine sodium carbonate, THF,water; (c) ethyl glycinate hydrochloride, Et₃N, EDCI, DCM; (d) arylaldehyde (R₂CHO), acetic anhydride, heat, 15 min; (e) amine form of R₃in (i) DMF, microwave heating or (ii) ethanol, room temperature;

FIG. 2 illustrates compounds of the disclosure;

FIG. 3A is a graph illustrating the dose-response curve of compound 1512on HCT-116 cell lines and C-166 cell line;

FIG. 3B is a graph illustrating the cytotoxic effect of 4112 againsthuman colon adenocarcinoma cancer cells (HCT-116, Caco-2 and HT-29) vs.normal baby hamster kidney cells (BHK). All cells were exposed todifferent concentrations of 4112 for 72 h. The cell viabilityconcentration curves were plotted, and the IC50 were determined.*denotes significance at P<0.001;

FIG. 4A illustrates the effect of 1512 at 32 μM (IC₅₀ concentration) onthe nuclear structures of HCT-116 cells compared to control (untreatedcells), where treated cells show nuclear condensation and fragmentationalong with the condensed blue fluorescence of4′,6′-diamidino-2-phenylindole (DAPI), and arrows point to apoptoticcells;

FIG. 4B illustrates morphological changes in HCT-116 cells followingexposure to compound 4112, where the cells were left untreated (control)or treated with the IC₅₀ for 24 and 48 h—the pictures at top show themorphological changes examined under light microscope and the images atthe bottom show the nuclei stained with DAPI and visualized underfluorescence microscope—arrows point to apoptotic cells;

FIG. 5 illustrates the effect of 4112 on HCT-116 cell cycle progression:Key LL=viable cells, LR=early apoptotic cells, UR=late apoptosis;

FIG. 6 illustrates the effect of 4112 on ROS production from HCT-116cells using DCFDA staining, with green fluorescence (middle)representing DCFDA staining, while DAPI (left) was used as counternuclear staining—scale bar of 100 μm; and

FIG. 7 is a graph illustrating the dose-response curve of HT-29 cellspheroids (expressed as % of vehicle treated cells) by increasingconcentration of 3712 (circle) and 4112 (triangle).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

Definitions

As used herein, the terms “compound”, “complex”, and “product” are usedinterchangeably, and are intended to refer to a chemical entity, whetherin the solid, liquid or gaseous phase, and whether in a crude mixture orpurified and isolated.

Throughout the specification and the appended claims, a given chemicalformula or name shall encompass all stereo and optical isomers andracemates thereof where such isomers exist. Unless otherwise indicated,all chiral (enantiomeric and diastereomeric) and racemic forms arewithin the scope of the disclosure. Many geometric isomers of C═C doublebonds, C═N double bonds, ring systems, and the like can also be presentin the compounds, and all such stable isomers are contemplated in thepresent disclosure. Cis- and trans- (or E- and Z-) geometric isomers ofthe compounds of the present disclosure are described and may beisolated as a mixture of isomers or as separated isomeric forms. Thepresent compounds can be isolated in optically active or racemic forms.Optically active forms may be prepared by resolution of racemic forms orby synthesis from optically active starting materials. All processesused to prepare compounds of the present disclosure and intermediatesmade therein are considered to be part of the present disclosure. Whenenantiomeric or diastereomeric products are prepared, they may beseparated by conventional methods, for example, by chromatography,fractional crystallization, or through the use of a chiral agent.Depending on the process conditions the end products of the presentdisclosure are obtained either in free (neutral) or salt form. Both thefree form and the salts of these end products are within the scope ofthe disclosure. If so desired, one form of a compound may be convertedinto another form. A free base or acid may be converted into a salt; asalt may be converted into the free compound or another salt; a mixtureof isomeric compounds of the present disclosure may be separated intothe individual isomers. Compounds of the present disclosure, free formand salts thereof, may exist in multiple tautomeric forms, in whichhydrogen atoms are transposed to other parts of the molecules and thechemical bonds between the atoms of the molecules are consequentlyrearranged. It should be understood that all tautomeric forms, insofaras they may exist, are included within the disclosure. Further, a givenchemical formula or name shall encompass all conformers, rotamers, orconformational isomers thereof where such isomers exist. Differentconformations can have different energies, can usually interconvert, andare very rarely isolatable. There are some molecules that can beisolated in several conformations. For example, atropisomers are isomersresulting from hindered rotation about single bonds where the stericstrain barrier to rotation is high enough to allow for the isolation ofthe conformers. It should be understood that all conformers, rotamers,or conformational isomer forms, insofar as they may exist, are includedwithin the present disclosure.

As used herein, the term “solvate” refers to a physical association of acompound of this disclosure with one or more solvent molecules, whetherorganic or inorganic. This physical association includes hydrogenbonding. In certain instances, the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. The solvent molecules in thesolvate may be present in a regular arrangement and/or a non-orderedarrangement. The solvate may comprise either a stoichiometric ornonstoichiometric amount of the solvent molecules. Solvate encompassesboth solution phase and isolable solvates. Exemplary solvents include,but are not limited to, water, methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, tert-butanol, ethyl acetate andother lower alkanols, glycerine, acetone, dichloromethane (DCM),dimethyl sulfoxide (DMSO), dimethyl acetate (DMA), dimethylformamide(DMF), isopropyl ether, acetonitrile, toluene, N-methylpyrrolidone(NMP), tetrahydrofuran (THF), tetrahydropyran, other cyclic mono-, di-and tri-ethers, polyalkylene glycols (e.g., polyethylene glycol,polypropylene glycol, propylene glycol), and mixtures thereof insuitable proportions. Exemplary solvates include, but are not limitedto, hydrates, ethanolates, methanolates, isopropanolates and mixturesthereof. Methods of solvation are generally known to those of ordinaryskill in the art.

As used herein, “pharmaceutically acceptable salt” refers to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic groups such as amines; and alkali or organic saltsof acidic groups such as carboxylic acids. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, andnitric; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like. The pharmaceutically acceptable salts of thepresent disclosure can be synthesized from the parent compound thatcontains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with a stoichiometric amount of the appropriatebase or acid in water or in an organic solvent, or in a mixture of thetwo; generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th Edition, MackPublishing Company, Easton, Pa. (1990), the disclosure of which ishereby incorporated by reference.

As used herein, the term “alkyl” unless otherwise specified refers toboth branched and straight chain aliphatic (non-aromatic) hydrocarbonswhich may be primary, secondary, and/or tertiary hydrocarbons typicallyhaving 1 to 32 carbon atoms (e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, etc.) and specifically includes, but is notlimited to, saturated alkyl groups such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,2-ethylhexyl, heptyl, octyl, nonyl, 3,7-dimethyloctyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, eicosyl, guerbet-type alkyl groups (e.g.,2-methylpentyl, 2-ethylhexyl, 2-proylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, 2-heptylundecyl, 2-octyldodecyl,2-nonyltridecyl, 2-decyltetradecyl, and 2-undecylpentadecyl), as well asunsaturated alkenyl and alkynyl variants such as vinyl, allyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,4-hexenyl, 5-hexenyl, oleyl, linoleyl, and the like.

The term “cycloalkyl” refers to cyclized alkyl groups. Exemplarycycloalkyl groups include, but are not limited to, saturated cycloalkylgroups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,norbornyl, and adamantyl, branched cycloalkyl groups such as1-methylcyclopropyl and 2-methylcyclopropyl, and cycloalkenyl groupssuch as cyclobutenyl, cyclopentenyl, and cyclohexenyl.

The term “aryl” means a carbocyclic aromatic monocyclic group containing6 carbon atoms which may be further fused to a second 5- or 6-memberedcarbocyclic group which may be aromatic, saturated or unsaturated. Arylincludes, but is not limited to, phenyl, anthracenyl, indanyl,1-naphthyl, 2-naphthyl, and tetrahydronaphthyl. The fused aryls may beconnected to another group either at a suitable position on thecycloalkyl/cycloalkenyl ring or the aromatic ring.

The term “arylalkyl”, as used herein, refers to a straight or branchedchain alkyl moiety (as defined above) that is substituted by an arylgroup (as defined above), examples of which include, but are not limitedto, benzyl, phenethyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl,2,4-dimethylbenzyl, 2-(4-ethylphenyl)ethyl, 3-(3-propylphenyl)propyl,and the like.

The term “alkoxy” refers to an —O-alkyl group. Example alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy (e.g.,n-propoxy and isopropoxy), and t-butoxy.

As used herein, the term “heterocycle” or “heterocyclyl” is intended tomean a stable 3-, 4-, 5-, 6-, or 7-membered monocyclic, 7-, 8-, 9-, 10-,or 11-membered bicyclic, or 7-, 8-, 9-, 10-, 11-, 12-, 13-, or14-membered polycyclic heterocyclic ring that is saturated, partiallyunsaturated, or fully unsaturated, and that contains carbon atoms and 1,2, 3 or 4 heteroatoms independently selected from the group consistingof N, O and S; and including any bicyclic or polycyclic group in whichany of the above-defined heterocyclic rings is fused to a carbocyclicring, the carbocyclic ring being either saturated, unsaturated, oraromatic (e.g., a benzene ring). The nitrogen and sulfur heteroatoms mayoptionally be oxidized (i.e., N→O and S(O)_(p), wherein p is 0, 1 or 2).The nitrogen atom may be substituted or unsubstituted (i.e., N or NRwherein R is H or another substituent, if defined). The heterocyclicring may be attached to its pendant group at any heteroatom or carbonatom that results in a stable structure. The heterocyclic ringsdescribed herein may be substituted on carbon or on a nitrogen atom ifthe resulting compound is stable. A nitrogen in the heterocycle mayoptionally be quaternized. It is preferred that when the total number ofS and O atoms in the heterocycle exceeds 1, then these heteroatoms arenot adjacent to one another. It is preferred that the total number of Sand O atoms in the heterocycle is not more than 1. When the term“heterocycle” is used, it is intended to include “heteroaryl” (whichwill be defined below).

Examples of heterocycles include, but are not limited to, acridinyl,azetidinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, imidazolopyridinyl, indolenyl,indolinyl, indolizinyl, indolyl (e.g., 1H-indolyl), isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isothiazolopyridinyl, isoxazolyl,isoxazolopyridinyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolopyridinyl, oxazolidinylperimidinyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,homopiperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolopyridinyl, pyrazolyl, pyridazinyl, pyridooxazolyl,pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl,pyrrolinyl, 2-pyrrolidonyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrazolyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thiazolopyridinyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl. Alsoincluded are fused ring and spiro compounds containing, for example, theabove heterocycles. Examples of 5- to 10-membered heterocycles include,but are not limited to, pyridinyl, thienyl, pyrrolyl, furyl, pyrazolyl,pyrazinyl, piperazinyl, piperidinyl, imidazolyl, imidazolidinyl,indolyl, tetrazolyl, isoxazolyl, morpholinyl, oxazolyl, oxadiazolyl,oxazolidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thiazolyl,triazinyl, triazolyl, benzimidazolyl, 1H-indazolyl, benzofuranyl,benzothiofuranyl, benztetrazolyl, benzo triazolyl, benzisoxazolyl,benzoxazolyl, oxindolyl, benzoxazolinyl, benzthiazolyl,benzisothiazolyl, isatinoyl, isoquinolinyl, octahydroisoquinolinyl,tetrahydroisoquinolinyl, tetrahydroquinolinyl, isoxazolopyridinyl,quinazolinyl, quinolinyl, isothiazolopyridinyl, thiazolopyridinyl,oxazolopyridinyl, imidazolopyridinyl, and pyrazolopyridinyl. Examples of5- to 6-membered heterocycles include, but are not limited to,pyridinyl, furyl, thienyl, pyrrolyl, pyrazolyl, pyrazinyl, piperazinyl,piperidinyl, imidazolyl, imidazolidinyl, indolyl, tetrazolyl,isoxazolyl, morpholinyl, oxazolyl, oxadiazolyl, oxazolidinyl,tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thiazolyl, triazinyl, andtriazolyl. Also included are fused ring and spiro compounds containing,for example, the above heterocycles. Examples of a bicyclic heterocyclicgroup include, but are not limited to, quinolinyl, isoquinolinyl,phthalazinyl, quinazolinyl, indolyl, isoindolyl, indolinyl,1H-indazolyl, benzimidazolyl, 1,2,3,4-tetrahydroquinolinyl,1,2,3,4-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydro-quinolinyl,2,3-dihydrobenzofuranyl, chromanyl, 1,2,3,4-tetrahydroquinoxalinyl, and1,2,3,4-tetrahydro-quinazolinyl.

The term “heteroaryl” is intended to mean stable monocyclic andpolycyclic aromatic hydrocarbons that include at least one heteroatomring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups areheterocyclyl groups which are aromatic, and may include, withoutlimitation, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl,indolyl (e.g., 1H-indolyl), pyrroyl, oxazolyl, benzofuryl, benzothienyl,benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl(e.g., 1H-indazolyl), 1,2,4-thiadiazolyl, isothiazolyl, purinyl,carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, andbenzodioxane. Heteroaryl groups may be substituted or unsubstituted. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or another substituent, if defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), whereinp is 0, 1 or 2).

The term “halo” or “halogen” includes fluoro, chloro, bromo and iodo.

As used herein, the term “substituted” refers to at least one hydrogenatom that is replaced with a non-hydrogen group, provided that normalvalencies are maintained and that the substitution results in a stablecompound. When a substituent is noted as “optionally substituted”, thesubstituent(s) are selected from alkyl, halo (e.g., chloro, bromo, iodo,fluoro), hydroxyl, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino(—NH₂), alkylamino (—NHalkyl), cycloalkylamino (—NHcycloalkyl),arylamino (—NHaryl), arylalkylamino (—NHarylalkyl), disubstituted amino(e.g., in which the two amino substituents are selected from alkyl, arylor arylalkyl, including substituted variants thereof, with specificmention being made to dimethylamino), alkanoylamino, aroylamino,arylalkanoylamino, thiol, alkylthio, arylthio, arylalkylthio,alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl,arylalkylsulfonyl, sulfonamide (e.g., —SO₂NH₂), substituted sulfonamide(e.g., —SO₂NHalkyl, —SO₂NHaryl, —SO₂NHarylalkyl, or cases where thereare two substituents on one nitrogen selected from alkyl, aryl, oralkylalkyl), nitro, cyano, carboxy, unsubstituted amide (i.e. —CONH₂),substituted amide (e.g., —CONHalkyl, —CONHaryl, —CONHarylalkyl or caseswhere there are two substituents on one nitrogen selected from alkyl,aryl, or alkylalkyl), alkoxycarbonyl, aryl, guanidine, heterocyclyl(e.g., pyridyl, furyl, morpholinyl, pyrrolidinyl, piperazinyl, indolyl,imidazolyl, thienyl, thiazolyl, pyrrolidyl, pyrimidyl, piperidinyl,homopiperazinyl), and mixtures thereof. The substituents may themselvesbe optionally substituted, and may be either unprotected, or protectedas necessary, as known to those skilled in the art, for example, astaught in Greene, et al., “Protective Groups in Organic Synthesis”, JohnWiley and Sons, Second Edition, 1991, hereby incorporated by referencein its entirety.

In cases wherein there are nitrogen atoms (e.g., amines) on compounds ofthe present disclosure, these may be converted to N-oxides by treatmentwith an oxidizing agent (e.g., mCPBA and/or hydrogen peroxides) toafford other compounds of this disclosure. Thus, shown and claimednitrogen atoms are considered to cover both the shown nitrogen and itsN-oxide (NO) derivative.

Compounds

In a first aspect, the present disclosure provides a compound of formula(I),

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof,

wherein:

R¹ is an optionally substituted aryl or an optionally substitutedheteroaryl;

R² is an optionally substituted aryl or an optionally substitutedheteroaryl; and

R³ is an optionally substituted alkylamino, an optionally substitutedcycloalkylamino, an optionally substituted arylamino, an optionallysubstituted heterocyclylamino, an optionally substituted heterocyclyl,and an optionally substituted dialkylamino.

In preferred embodiments, the double bond formed between the enaminefunctionality and the R² substituent adopts a Z-configuration, that is,the enamine nitrogen and the R² substituent reside on the same side ofthe double bond. The double bond formed between the amide and the R¹substituent may adopt either an E- or Z-configuration, preferably anE-configuration. That is, it is preferred that the amide functionalgroup and the R¹ substituent reside on opposite sides of the doublebond.

In some embodiments, R¹ is an aromatic moiety (e.g., aryl or heteroaryl)with a direct bond between the aromatic moiety and the double bond towhich it is attached (such that the double bond is in conjugation withthe aromatic group of R¹), thereby forming a cinnamamide-type moiety ora heteroarylacrylamide-type moiety. R¹ may be an optionally substitutedmonocyclic aryl or heteroaryl group or an optionally substitutedbicyclic aryl or heteroaryl group, preferably R¹ is an optionallysubstituted monocyclic aryl or heteroaryl group. In some embodiments, R¹may contain anywhere from 4 to 10 total carbon atoms, including anycarbon atoms of substituents present, preferably 4 to 8 total carbonatoms, preferably 4 to 7 carbon atoms.

When R¹ is an optionally substituted heteroaryl group, it is preferredthat R¹ is a sulfur-containing heteroaryl substituent, for example,thienyl, benzothienyl, benzisothiazolyl, isothiazolyl,isothiazolopyridinyl, benzthiazolyl, phenothiazinyl, thiazolyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl), thiazolopyridinyl,thienothiazolyl, thienooxazolyl, and thienoimidazolyl, just to name afew. Preferably, R¹ is an unsubstituted heteroaryl group, with specificmention being made to thienyl, preferably 2-thienyl.

When R¹ is an optionally substituted aryl group, it is preferred that R¹is phenyl (unsubstituted) or a substituted phenyl group. When R¹ is asubstituted phenyl group, the phenyl moiety is preferably substitutedwith one or more substituents selected from a halo, preferably chloro orfluoro, preferably a chloro; an alkoxy, preferably a C₁ to C₄ alkoxy,preferably a C₁ to C₃ alkoxy, preferably a C₁ to C₂ alkoxy (e.g.,methoxy or ethoxy), preferably a C₁ alkoxy (e.g., methoxy); and analkyl, preferably a C₁ to C₄ alkyl, preferably a C₁ to C₃ alkyl,preferably a C₁ to C₂ alkyl (e.g., methyl or ethyl), preferably a C₁alkyl (e.g., methyl). In preferred embodiments, when R¹ is a substitutedphenyl, the phenyl group is substituted (at least) in the 4-position(para to the connection to the rest of the compound of formula (I)),preferably the phenyl group contains a single substituent at the4-position, and R¹ is a 4-halophenyl, a 4-alkoxyphenyl, or4-alkylphenyl, preferably 4-chlorophenyl, 4-methoxyphenyl, or4-methylphenyl.

In some embodiments, R² is an aromatic moiety (e.g., aryl or heteroaryl)with a direct bond between the aromatic moiety and the double bond towhich it is attached, such that the double bond is in conjugation withthe aromatic group of R². In some embodiments, R² may be an optionallysubstituted monocyclic aryl or heteroaryl group or an optionallysubstituted bicyclic aryl or heteroaryl group, preferably R² is anoptionally substituted monocyclic aryl group or an optionallysubstituted bicyclic heteroaryl group. In some embodiments, R² maycontain anywhere from 5 to 10 total carbon atoms, including any carbonatoms of substituents present, preferably 6 to 8 total carbon atoms,preferably 7 to 8 carbon atoms.

When R² is an optionally substituted heteroaryl group, it is preferredthat R² is a nitrogen-containing heteroaryl substituent, for example,pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,quinolyl, isoquinolyl, imidazolyl, indolyl (e.g., 1H-indolyl), pyrroyl,oxazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl (e.g.,1H-indazolyl), 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl,and benzimidazolyl, just to name a few.

In some embodiments, R² is an optionally substituted indolyl or pyridyl,preferably a 1H-indolyl or 3-pyridyl. Preferably, R² is an unsubstitutedheteroaryl group. In preferred embodiments, R² is a nitrogen-containingheteroaryl substituent having a protic —NH— group, preferably a bicyclicnitrogen-containing heteroaryl substituent having a protic —NH— group,with specific mention being made to indolyl (e.g., 1H-indolyl),benzimidazole, and indazolyl (e.g., 1H-indazolyl), preferably indolyl.In preferred embodiments, R² is 1H-indol-3-yl.

When R² is an optionally substituted aryl group, it is preferred that R²is phenyl (unsubstituted) or a substituted phenyl group. When R² is asubstituted phenyl group, the phenyl moiety is preferably substitutedwith one or more substituents selected from a halo, preferably chloro orfluoro, preferably fluoro; a dialkylamino, preferably a di-(C₁ to C₄alkyl)amino, preferably a di-(C₁ to C₃ alkyl)amino, preferably a di-(C₁to C₂ alkyl)amino, preferably a di-(C₁ alkyl)amino (e.g.,dimethylamino); an alkoxy, preferably a C₁ to C₃ alkoxy, preferably a C₁to C₂ alkoxy (e.g., methoxy or ethoxy), preferably a C₁ alkoxy (e.g.,methoxy); a hydroxy; and a nitro. In preferred embodiments, when R² is asubstituted phenyl, the phenyl group is mono- or di-substituted, andpreferably is substituted (at least) in the 4-position (para to theconnection to the rest of the compound of formula (I)). In preferredembodiments, R² is a 4-halophenyl, a 4-dialkylaminophenyl,3-alkoxy-4-hydroxyphenyl, or a 4-nitrophenyl, preferably 4-fluorophenyl,4-dimethylaminophenyl, 3-methoxy-4-hydroxyphenyl, or 4-nitrophenyl.

R³ may an optionally substituted alkylamino (—NHalkyl), an optionallysubstituted cycloalkylamino (—NHcycloalkyl), an optionally substitutedacylamino (—NHaryl), an optionally substituted heterocyclylamino(—NH-heterocyclyl, where a nitrogen atom which is not part of theheterocyclic ring is bonded to the carbonyl group of the compound offormula (I)), an optionally substituted heterocyclyl (-heterocyclyl,where a heterocyclic ring nitrogen is directly bonded the carbonyl groupof the compound of formula (I)), and an optionally substituteddialkylamino (—N(alkyl)(alkyl)).

In preferred embodiments, R³ represents an amino group which is notlocated within a ring and which is directly bonded to one or morealiphatic hydrocarbon moieties. In some embodiments, R³ is anunsubstituted C₁ to C₄ alkylamino, for example, ethylamino,n-propylamino, n-butylamino, and sec-butylamino, preferably a C₂ to C₄alkylamino, preferably a C₃ to C₄ alkylamino, preferably a C₃alkylamino. In preferred embodiments, R³ is a linear unsubstituted C₁ toC₄ alkylamino, preferably n-propylamino (—NHCH₂CH₂CH₃). In someembodiments, R³ is a substituted C₁ to C₄ alkylamino, for example, a C₂to C₄ alkylamino in which the alkyl group is substituted with a hydroxy,an alkoxy, or a heterocyclyl group, with specific mention being made tomorpholinyl, and furyl substituents (e.g., where R³ is furfurylamino or2-morpholinoethylamino). In some embodiments, R³ is an optionallysubstituted C₅ to C₁₀ cycloalkylamino, preferably C₅ to C₈cycloalkylamino, preferably C₅ to C₆ cycloalkylamino, for examplecyclopentylamino, cyclohexylamino, and 1-adamantylamino, preferablycyclopentylamino (—NHC₅H₉). In some embodiments, R³ is an optionallysubstituted di-(C₁ to C₃ alkyl)amino, where each alkyl group attached tothe amino group can be the same or different, preferably different.Preferably, R³ is a di-(C₁ to C₂ alkyl)amino, with specific mentionbeing made to methylethylamino (—N(Me)Et). Most preferably, R³ is anon-aromatic amino group such as an alkylamino, a cycloalkylamino, or adialkylamino group which is either unsubstituted or which is substitutedwith non-aromatic substituents.

Alternatively, R³ may be an amino group which is directly bonded to anaromatic moiety such as an aryl or heteroaryl group (R³ is an arylaminogroup or a heteroarylamino group) or another heteroatom or may be anamino group in which the nitrogen that is bonded to the carbonyl groupof the compound of formula (I) is present within a ring (e.g., thenitrogen is a ring nitrogen of a heterocyclic ring). For example, R³ maybe an optionally substituted phenylamino (e.g., phenylamino,3-cyanophenylamino, 4-fluorophenylamino), an optionally substitutedpyridylamino, an optionally substituted N-piperazinylamino (R³ is a1-piperazinylamino), an optionally substituted morpholinyl, or anoptionally substituted pyrrolidinyl,

In some embodiments, R³ is selected from the group consisting ofcyclopentylamino, 1-adamantylamino, 3-pyridylamino,4-methyl-1-piperazinylamino, ethylamino, n-propylamino, n-butylamino,sec-butylamino, 3-cyanophenylamino, 4-fluorophenylamino, furfurylamino,2-morpholinoethylamino, 2-hydroxyethylamino, 4-morpholinyl,1-pyrrolidinyl, and methylethylamino. Most preferably, R³ isn-propylamino.

The compound of formula (I) may be selected from the group consisting of

In preferred embodiments, the compound of formula (I) is selected fromthe group consisting of

The compounds of the present disclosure may be prepared by methods knownto those of ordinary skills in the art. The following methods set forthbelow are provided for illustrative purposes and are not intended tolimit the scope of the disclosure.

The compounds of formula (I) may, for example, be synthesized accordingto a Erlenmeyer-Plöchl azlactone synthetic route illustrated in FIG. 1C.Briefly, the compounds may be formed starting from an aldehyde offormula R¹CHO, which may be subjected to a condensation reaction, forexample using malonic acid, an organic base (e.g., pyridine), and heat,to form a cinnamic acid compound of formula (II),

wherein R¹ is as defined previously.

The cinnamic acid compound of formula (II) may be subjected to amidationwith glycine, or a salt or an ester thereof (e.g., ethylglycinate) usingany known peptide bond forming technique known to those of ordinaryskill in the art to form cinnamoylglycine analogs of formula (III)

wherein R^(a) is hydrogen (formula III is cinnamoylglycine type) or analkyl group (formula III is cinnamoylglycinate ester type). Such peptidebond forming reactions may be accomplished using reagents including, butnot limited to, carbodiimides [e.g., dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or EDCI),N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide-methyl-p-toluenesulfonate(CMC)], triazolopyridines and salts thereof [e.g.,1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU), 7-aza-1-hydroxybenzotriazole (HOAt)],benzotriazoles and salts thereof [e.g.,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), (benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 1-hydroxybenzotriazole (HOBt)], benzotriazin-4-ones [e.g.,3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one],carbonyldiimidazole (CDI), phosgene, triphosgene, benzoates (e.g.,methyl benzoate), chloroformates [e.g., butyl-, t-butyl-, isobutyl-,isopropyl-, and 4-nitrophenyl chloroformate], cyanuric chlorides andcyanuric fluorides including derivatives of cyanuric chlorides/fluorides[e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT),4-(4,6-dimethoxy-[1,3,5]triazin-2-yl)-4-methylmorpholin-4-ium chloride(DMTMM)], N-hydroxyphthalimide, aminopyridines (e.g.,4-(dimethylamino)pyridine), thionyl chloride, oxalyl chloride,phosphorous pentachloride, bis(2-methoxyethyl)aminosulfur trifluoride,diethylaminosulfur trifluoride, and propylphosphonic anhydride (T3P).Preferably, the cinnamoylglycine analogs of formula (III) are preparedusing the consecutive addition of oxalyl chloride and glycine or a saltthereof, or alternatively using a peptide coupling reagent such as EDCIand ethyl glycinate or a salt thereof.

Cyclocondensation of the cinnamoylglycine analogs of formula (III) maynext be effected under Erlenmeyer conditions, for example, heating thecinnamoylglycine analogs of formula (III) in the presence of an aldehydeof formula R²CHO and a carboxylic acid anhydride (e.g., aceticanhydride), to form an azlactone (also known as an oxazolone) of formulaIV

wherein R¹ and R² are as defined previously, and the newly formed doublebond adopts a Z-configuration where the azlactone ring nitrogen and theR² substituent reside on the same side of the double bond.

The azlactone of formula IV may then be reacted with a variety ofaliphatic and aromatic amines (the amine form of R³ whereby anucleophilic amine group present in R³ reacts with the azlactonecarbonyl group) to form the compounds of formula (I). In general,reactive amines such as aliphatic and benzylic amines may react with theazlactone of formula IV under room temperature conditions, for examplein an alcoholic solvent such as methanol, ethanol, propanol,isopropanol, n-butanol, isobutanol, n-pentanol, n-hexanol,3-methyl-3-buten-1-ol, ethylene glycol, diethylene glycol, triethyleneglycol, tetraethylene glycol, propylene glycol, dipropylene glycol,1,3-propanediol, etc., while less reactive amines such as aromaticamines may be reacted with the azlactone of formula IV using heat (e.g.,microwave heat) and high boiling solvents such as dimethylformamide.

The progress of any such reactions may be monitored by methods known tothose of ordinary skill in the art, such as thin layer chromatography,gas chromatography, nuclear magnetic resonance, infrared spectroscopy,and high pressure liquid chromatography combined with ultravioletdetection or mass spectroscopy. The compounds of formula (I) may beisolated and purified by methods known to those of ordinary skill in theart, such as one or more of crystallization, precipitation, filtration,solvent evaporation, aqueous work-up, solvent extraction, drying,distillation, column chromatography, high pressure liquid chromatography(HPLC), lyophilization, and the like.

Of course, it should be understood that the compounds of formula (I) maybe synthesized through various other synthetic schemes, reactions typesand conditions, and isolation/purification procedures and still beconsidered a part of the present disclosure.

Pharmaceutical Compositions

According to a second aspect, the present disclosure relates to apharmaceutical composition which comprises a therapeutically effectiveamount of one or more of the compounds of formula (I), formulatedtogether with one or more pharmaceutically acceptable carriers and/orexcipients, and optionally, one or more additional therapeutic agents.As described in detail below, the pharmaceutical compositions of thepresent disclosure may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for buccal,sublingual, and systemic absorption, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained release formulation; (3) topical application, for example, asa cream, ointment, or a controlled release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, a “composition” or a “pharmaceutical composition” refersto a mixture of an active ingredient(s) with other chemical components,such as pharmaceutically acceptable carriers and/or excipients. Onepurpose of a composition is to facilitate administration of thecompounds disclosed herein in any of its embodiments to a subject.Pharmaceutical compositions of the present disclosure may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.Depending on the intended mode of administration (e.g., oral,parenteral, or topical), the composition can be in the form of solid,semi-solid or liquid dosage forms, such as tablets, suppositories,pills, capsules, powders, liquids, or suspensions, preferably in unitdosage form suitable for single administration of a precise dosage.

The term “active ingredient” or “active compound”, as used herein,refers to an ingredient in the composition that is biologically active,for example, one or more compounds represented by formula (I), a saltthereof, a solvate thereof, a tautomer thereof, a stereoisomer thereof,or any mixtures thereof. In some embodiments, additional therapeuticagents, in addition to the compound of the current disclosure, may beincorporated into a pharmaceutical composition, for example, a secondactive ingredient which is chemically distinct from the compounds offormula (I).

When the compounds of formula (I) are administered as pharmaceuticals,to humans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1 to 99 wt. % of activeingredient(s) in combination with a pharmaceutically acceptable carrierand/or excipient. In some embodiments, the pharmaceutical compositioncomprises 0.01 to 99 wt. % of the compound of formula (I) relative to atotal weight of the pharmaceutical composition. For example, thepharmaceutical composition may contain at least 0.01 wt. %, at least0.05 wt. %, at least 0.1 wt. %, at least 0.5 wt. %, at least 5 wt. %, atleast 10 wt. %, at least 15 wt. %, at least 20 wt. %, at least 25 wt. %,at least 30 wt. %, at least 35 wt. %, at least 40 wt. %, at least 45 wt.%, at least 50 wt. %, at least 55 wt. %, at least 60 wt. %, at least 65wt. %, at least 70 wt. %, and up to 99 wt. %, up to 98 wt. %, up to 95wt. %, up to 90 wt. %, up to 85 wt. %, up to 80 wt. %, up to 75 wt. % ofthe compound of formula (I) relative to a total weight of thepharmaceutical composition. In preferred embodiments, the pharmaceuticalcomposition includes 5 to 15 wt. %, preferably 6 to 14 wt. %, preferably7 to 13 wt. %, preferably 8 to 12 wt. %, preferably 9 to 11 wt. %,preferably 10 wt. % of the compound of formula (I), based on a totalweight of the pharmaceutical composition.

In some embodiments, the active ingredient of the current disclosure,e.g., the compound of formula (I), a salt thereof, a solvate thereof, atautomer thereof, a stereoisomer thereof, or any mixtures thereof, mayprovide utility as an anticancer agent in reducing the viability ofcancer cells derived from human cancer cell lines including, but notlimited to, breast cancer cell lines (e.g., MDA-MB-231, MCF-7, SK-BR-3,T47D, VP303); stomach cancer cell lines (e.g., N87, SNU-16, SNU-5,SNU-1, KATO III, AGS); colon/colorectal cancer cell lines (e.g.,HCT-116, CACO-2, HT-29, HCT15, MDST8, GPSd, DLD1, SW620, SW403, T84);leukemia cell lines (e.g., HL-60, CESS, CCRF-CEM, CEM/C1, KASUMI-1,ARH-77); liver cancer cell lines (e.g., HepG2, PLC/PRF/5, THLE-3, C3A,SNU-182, SNU-398, SNU-387, SNU-423, SNU-475, SNU-449, and Hep 3B2.1-7);lung cancer cell lines (e.g., A549, NCI-H460, SHP-77, COR-L23/R,NCI-H69/LX20); brain tumor cell lines (e.g., U251); ovarian cancer celllines (e.g., NCI-ADR/RES, OVCAR-03, A2780, A2780cis, OV7, PEO23);prostate cancer cell lines (e.g., PC-3); renal cancer cell lines (e.g.,786-0); and skin cancer or melanoma cell lines (e.g., UACC-62, C32TG,A375, MCC26). Preferably, the active ingredient of the currentdisclosure, e.g., the compound of formula (I), a salt thereof, a solvatethereof, a tautomer thereof, a stereoisomer thereof, or any mixturesthereof, provides utility as an anticancer agent in reducing theviability of cancer cells derived from human colon/colorectal cancercell lines (e.g., HCT-116, CACO-2, HT-29).

In some embodiments, the cancer cells are collected from a human patientwho is at risk of having, is suspected of having, has been diagnosedwith, or is being monitored for recurrence of at least one type ofcancer, preferably colon/colorectal cancer.

In some embodiments, the ability of the active ingredient to reduce theviability of cancer cells may be determined by contacting thepharmaceutical composition with the cancer cells and then performingcell viability assays. Methods of such assays include, but are notlimited to, sulforhodamine-B (SRB) assay, ATP test, Calcein AM assay,clonogenic assay, ethidium homodimer assay, Evans blue assay,2′,7′-dichlorofluorescin diacetate (DCFDA) staining assay, fluoresceindiacetate hydrolysis/propidium iodide staining assay, annexinV/fluorescein isothiocyanate (FITC)/propidium iodide staining assay,flow cytometry, Formazan-based assays (MTT, XTT), green fluorescentprotein assay, lactate dehydrogenase (LDH) assay, methyl violet assay,propidium iodide assay, Resazurin assay, trypan blue assay,4′,6′-diamidino-2-phenylindole (DAPI) assay, TUNEL assay, andprimary(1°) colonosphere formation assay. In a preferred embodiment, aMTT assay is used. In another preferred embodiment, a Resazurin assay isused.

As is well understood in the art, the IC₅₀ value of a compound/mixtureis a concentration of that compound/mixture which causes the death of50% of the cellular population to which the compound/mixture is added.In some embodiments, the IC₅₀ of the compound of formula (I), the saltthereof, the solvate thereof, the tautomer thereof, the stereoisomerthereof, or the mixture thereof against colorectal cancer cells, forexample HCT-116, CACO-2, and HT-29 human cancer cell lines, is less than200 μM, preferably less than 150 μM, preferably less than 100 μM,preferably less than 90 μM, preferably less than 80 μM, preferably lessthan 70 μM, preferably less than 60 μM, preferably less than 50 μM,preferably less than 40 μM, preferably less than 30 μM, preferably lessthan 25 μM, preferably less than 20 μM, preferably less than 15 μM,preferably less than 10 μM, preferably less than 5 μM, preferably lessthan 4 μM, preferably less than 3 μM, preferably less than 2 μM,preferably less than 1 μM, for example, from 0.8 to 20 μM, preferablyfrom 0.9 to 15 μM, preferably from 1 to 10 μM, preferably from 1.5 to 8μM, preferably from 2 to 6 μM.

In some embodiments, additional therapeutic agents in addition to thecompound of the current disclosure may be incorporated into thepharmaceutical composition. In some embodiments, the pharmaceuticalcomposition includes an additional therapeutic agent that is chemicallydistinct from the compound of formula (I), such as a chemotherapeuticagent or an anticancer agent, for the treatment or prevention ofneoplasm, of tumor or cancer cell division, growth, proliferation and/ormetastasis in the subject; induction of death or apoptosis of tumorand/or cancer cells; and/or any other forms of proliferative disorder.

The additional therapeutic agent may be an anticancer agent and mayinclude, but is not limited to, at least one of a mitotic inhibitor; analkylating agent; an antimetabolite; a cell cycle inhibitor; an enzyme;a topoisomerase inhibitor; a biological response modifier; ananti-hormone; a tubulin inhibitor; a tyrosine-kinase inhibitor; anantiangiogenic agent such as MMP-2, MMP-9 and COX-2 inhibitor; ananti-androgen; a platinum coordination complex (oxaliplatin,carboplatin); a substituted urea such as hydroxyurea; a methylhydrazinederivative; an adrenocortical suppressant, e.g., mitotane,aminoglutethimide; a hormone and/or hormone antagonist such as theadrenocorticosteriods (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate), an estrogen (e.g., diethylstilbestrol);an antiestrogen such as tamoxifen; androgen, e.g., testosteronepropionate; and an aromatase inhibitor, such as anastrozole, andAROMASIN (exemestane).

Exemplary additional therapeutic agents include, but are not limited to,tubulin binding agents including paclitaxel, epothilone, docetaxel,discodermolide, etoposide, vinblastine, vincristine, teniposide,vinorelbine, and vindesine; tyrosine-kinase inhibitors includingimatinib, nilotinib, dasatinib, bosutinib, ponatinib, and bafetinib;alkylating antineoplastic agents including busulfan, carmustine,chlorambucil, cyclophosphamide, cyclophosphamide, dacarbazine,ifosfamide, lomustine, mechlorethamine, melphalan, mercaptopurine,procarbazine; antimetabolites including cladribine, cytarabine,fludarabine, gemcitabine, pentostatin, 5-fluorouracil, clofarabine,capecitabine, methotrexate, thioguanine; cytotoxic antibiotics includingdaunorubicin, doxorubicin, idarubicin, mitomycin, actinomycin,epirubicin; topoisomerase inhibitors including irinotecan, mitoxantrone,topotecan, and mixtures thereof.

As used herein, the phrase “pharmaceutically acceptable carrier and/orexcipient” means a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, carrier, diluent, excipient,manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), or solvent encapsulating material, involvedin carrying or transporting the subject compound from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, com oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) pH buffered solutions; (21) polyesters,polycarbonates and/or polyanhydrides; and (22) other non-toxiccompatible substances employed in pharmaceutical formulations, such ascyclodextrins, liposomes, and micelle forming agents, e.g., bile acids,just to name a few.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the pharmaceuticalcompositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Methods of preparing these pharmaceutical compositions include the stepof bringing into association a compound of formula (I) with thepharmaceutically acceptable carrier and/or excipient, and, optionally,one or more accessory ingredients. In general, the compositions areprepared by uniformly and intimately bringing into association acompound of the present disclosure with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Pharmaceutical compositions of the present disclosure suitable for oraladministration may be in the form of capsules, cachets, pills, tablets,lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofa compound of formula (I) as an active ingredient. A compound of formula(I) of the present disclosure may also be administered as a bolus,electuary or paste.

In solid dosage forms of the present disclosure for oral administration(capsules, tablets, pills, dragees, powders, granules, troches and thelike), the active ingredient is mixed with one or more pharmaceuticallyacceptable carriers and/or excipients, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants (e.g., fatty acidesters of sorbitan and polyalkolyated fatty acid esters of sorbitan suchas TWEEN 80, available from Sigma-Aldrich); (8) absorbents, such askaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets, and other soliddosage forms of the pharmaceutical compositions of the presentdisclosure may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above described excipients.

Liquid dosage forms for oral administration of the compounds of thedisclosure include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, com, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters (including polyoxyethylene fatty acidesters of sorbitan, e.g., TWEEN 80), microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar and tragacanth, andmixtures thereof. In preferred embodiments, the pharmaceuticalcomposition is in the form of a suspension, comprising, consisting of,or consisting essentially of the compound of formula (I) and thepharmaceutically acceptable carrier and/or excipient, which ispreferably a suspending agent (preferably a polyoxyethylene sorbitanester, preferably a polyoxyethylene fatty acid ester of sorbitan, e.g.,TWEEN 80) in an inert diluent (preferably water). Preferably the contentof the suspending agent in the suspension ranges from 0.01 to 1 wt. %,preferably 0.05 to 0.8 wt. %, preferably 0.1 to 0.6 wt. %, preferably0.5 wt. %, based on a total weight of the suspension.

Formulations of the pharmaceutical compositions of the disclosure forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of formula (I)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound(s).

Formulations of the pharmaceutical compositions which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this disclosure include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this disclosure, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisdisclosure, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present disclosure to the body. Suchdosage forms can be made by dissolving or dispersing the compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this disclosure.

Pharmaceutical compositions of this disclosure suitable for parenteraladministration comprise one or more compounds of formula (I) incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants (e.g., TWEEN 80).

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the pharmaceutical compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsuled matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly (orthoesters) and poly (anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

In some embodiments, the pharmaceutical composition contains 1 to 99.9wt. %, preferably 5 to 95 wt. %, preferably 10 to 90 wt. %, preferably15 to 85 wt. %, preferably 20 to 80 wt. %, preferably 30 to 75 wt. %,preferably 40 to 70 wt. %, preferably 50 to 65 wt. % of thepharmaceutically acceptable carrier and/or excipient, relative to atotal weight of the pharmaceutical composition.

Therapeutic Applications and Methods

According to a third aspect, the present disclosure relates to a methodfor treating a proliferative disorder. The method involves administeringa therapeutically effective amount of one or more compounds of formula(I) per se, or a pharmaceutical composition of the second aspect to asubject.

In some embodiments, the proliferative disorder is cancer. Types ofcancers that may be treated with the compounds of this disclosureinclude, but are not limited to, brain cancers, skin cancers, bladdercancers, ovarian cancers, breast cancers, gastric cancers, pancreaticcancers, prostate cancers, colon/colorectal cancers, blood cancers, lungcancers and bone cancers. In some embodiments, the compounds of thisdisclosure can be used for the treatment of any cancer type that failsto undergo apoptosis in a patient. This includes, but is not limited to:solid tumors, including but not limited to carcinomas; sarcomasincluding Kaposi's sarcoma; erythroblastoma; glioblastoma; meningioma;astrocytoma; melanoma; and myoblastoma. Treatment or prevention ofnon-solid tumor cancers, such as leukemia, is also contemplated by thisinvention.

Examples of such cancer types include neuroblastoma, intestine carcinomasuch as rectum carcinoma, colon carcinoma, familiar adenomatouspolyposis carcinoma and hereditary non-polyposis colorectal cancer,esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynxcarcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma,adenocarcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, renal carcinoma, kidney parenchymal carcinoma, ovariancarcinoma, cervix carcinoma, uterine corpus carcinoma, endometriumcarcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma,testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, braintumors such as glioblastoma, astrocytoma, meningioma, medulloblastomaand peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkinlymphoma, Bur-kitt lymphoma, acute lymphatic leukemia (ALL), chroniclymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloidleukemia (CML), adult T-cell leukemia lymphoma, diffuse laige B-celllymphoma (DLBCL), hepatocellular carcinoma, gall bladder carcinoma,bronchial carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroid melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma and plasmocytoma. In preferred embodiments, the cancer iscolon or colorectal cancer.

As used herein, the terms “treat”, “treatment”, and “treating” in thecontext of the administration of a therapy to a subject in need thereofrefers to the reduction or inhibition of the progression and/or durationof a disease (e.g., cancer), the reduction or amelioration of theseverity of the disease, the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies,preventing the disease from occurring in a subject that may bepredisposed to the disease but does not yet experience or exhibitsymptoms of the disease (prophylactic treatment), slowing or arrestingdisease development, ameliorating the disease, providing relief from thesymptoms or side-effects of the disease (including palliativetreatment), and causing regression of the disease. Specific to cancer,and in particular colon or colorectal cancer, these terms may refer to:(1) a stabilization, reduction (e.g., by more than 10%, 20%, 30%, 40%,50%, preferably by more than 60% of the population of cancer cellsand/or tumor size before administration), or elimination of the cancercells, (2) inhibiting cancerous cell division and/or cancerous cellproliferation, (3) relieving to some extent (or, preferably,eliminating) one or more symptoms associated with a pathology related toor caused in part by unregulated or aberrant cellular division, (4) anincrease in disease-free, relapse-free, progression-free, and/or overallsurvival, duration, or rate, (5) a decrease in hospitalization rate, (6)a decrease in hospitalization length, (7) eradication, removal, orcontrol of primary, regional and/or metastatic cancer, (8) astabilization or reduction (e.g., by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, preferably at least 80% relative to the initial growth rate)in the growth of a tumor or neoplasm, (9) an impairment in the formationof a tumor, (10) a reduction in mortality, (11) an increase in theresponse rate, the durability of response, or number of patients whorespond or are in remission, (12) the size of the tumor is maintainedand does not increase or increases by less than 10%, preferably lessthan 5%, preferably less than 4%, preferably less than 2%, (13) adecrease in the need for surgery (e.g., colectomy, mastectomy), and (14)preventing or reducing (e.g., by more than 10%, more than 30%,preferably by more than 60% of the population of metastasized cancercells before administration) the metastasis of cancer cells.

The term “subject” and “patient” are used interchangeably. As usedherein, they refer to any subject for whom or which therapy, includingwith the compositions according to the present disclosure is desired. Inmost embodiments, the subject is a mammal, including but not limited toa human, a non-human primate such as a chimpanzee, a domestic livestocksuch as a cattle, a horse, a swine, a pet animal such as a dog, a cat,and a rabbit, and a laboratory subject such as a rodent, e.g., a rat, amouse, and a guinea pig. In preferred embodiments, the subject is ahuman.

The subject may be any subject already with the disease, a subject whichdoes not yet experience or exhibit symptoms of the disease, or a subjectpredisposed to the disease. In preferred embodiments, the subject is aperson who is predisposed to cancer, e.g., a person with a familyhistory of cancer. Women who have (i) certain inherited genes (e.g.,mutated BRCA1 and/or mutated BRCA2), (ii) been taking estrogen alone(without progesterone) after menopause for many years (at least 5, atleast 7, or at least 10), and/or (iii) been taking fertility drugclomiphene citrate, are at a higher risk of contracting breast cancer.People who (i) consumes a diet high in salty and smoked foods and/or lowin fruits and vegetables, (ii) had infection with Helicobacter pylori,and/or (iii) long-term stomach inflammation are at a higher risk ofcontracting stomach cancer. People who (i) had chemotherapy andradiation therapy for other cancers, (ii) has genetic disorders, such asDown syndrome, and/or (iii) exposure to certain chemicals, such asbenzene are at a higher risk of contracting leukemia. People who (i) hadinflammatory bowel disease, or a genetic syndrome such as familialadenomatous polyposis (FAP) and hereditary non-polyposis colorectalcancer (Lynch syndrome), and/or (ii) consumes a low-fiber and high-fatdiet are at a higher risk of contracting colon cancer. Any subject withsuch predispositions, in combination with sound medical judgment, may becandidates for the treatment methods described herein.

In some embodiments, the subject has leukemia, stomach, colon, and/orbreast cancer and is currently undergoing, or has completed one or morechemotherapy regimens. In some embodiments, the subject has beenpreviously administered/treated with, or is being currentlyadministered/treated with, a thymidylate synthase inhibitor (e.g.,capecitabine, fluorouracil (5-FU)), a thymidine phosphorylase (TPase)inhibitor (e.g., tipiracil, trifluridine), topoisomerase I inhibitor(e.g., irinotecan), a DNA synthesis inhibitor (e.g., oxaliplatin),and/or a targeted therapy (e.g., cetuximab, bevacizumab, panitumumab,zivaflibercept, ramucirumab). In some embodiments, the subject has beenpreviously administered/treated with, or is being currentlyadministered/treated with, a tubulin binding drug such as paclitaxel,epothilone, docetaxel, discodermolide, etoposide, vinblastine,vincristine, teniposide, vinorelbine, and vindesine, and developedresistance to the tubulin binding drug. In some embodiments, the subjecthas been previously administered/treated with, or is being currentlyadministered/treated with, a tyrosine-kinase inhibitor such as imatinib,nilotinib, dasatinib, bosutinib, ponatinib, and bafetinib, and developeddrug resistance via (i) Bcr-Abl dependent mechanisms involving Bcr-Ablduplication, Bcr-Abl mutation, T3151 mutation, and/or P-loop mutations,or (ii) Bcr-Abl Independent mechanisms involving drug efflux caused byP-glycoproteins, drug import by organic cation transporter 1, and/oralternative signaling pathway activation.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to the methods that may be used to enable deliveryof the active ingredient and/or the pharmaceutical composition to thedesired site of biological action. Routes or modes of administration areas set forth herein. These methods include, but are not limited to, oralroutes, intraduodenal routes, parenteral injection (includingintravenous, subcutaneous, intraperitoneal, intramuscular,intravascular, or infusion), topical and rectal administration. Those ofordinary skill in the art are familiar with administration techniquesthat can be employed. In preferred embodiments, the active ingredient(e.g., the compound of formula (I)) or the pharmaceutical compositiondescribed herein are administered orally, preferably as an oralsuspension.

The dosage amount and treatment duration are dependent on factors, suchas bioavailability of a drug, administration mode, toxicity of a drug,gender, age, lifestyle, body weight, the use of other drugs and dietarysupplements, the disease stage, tolerance and resistance of the body tothe administered drug, etc., and then determined and adjustedaccordingly. The terms “effective amount”, “therapeutically effectiveamount”, or “pharmaceutically effective amount” refer to that amount ofthe active ingredient being administered which will relieve to someextent one or more of the symptoms of the disease being treated. Theresult can be a reduction and/or alleviation of the signs, symptoms, orcauses of a disease, or any other desired alteration of a biologicalsystem. An appropriate “effective amount” may differ from one individualto another. An appropriate “effective amount” in any individual case maybe determined using techniques, such as a dose escalation study.Typically, an effective amount of the compound of formula (I) to treatcancers such as colon or colorectal cancer, in terms of mg of thecompound of formula (I) per body weight of the subject (kg), ranges from0.1 to 2,000 mg/kg, preferably 1 to 1,500 mg/kg, preferably 5 to 1,000mg/kg, preferably 10 to 900 mg/kg, preferably 15 to 800 mg/kg,preferably 20 to 700 mg/kg, preferably 30 to 600 mg/kg, preferably 40 to500 mg/kg, preferably 50 to 400 mg/kg, preferably 60 to 300 mg/kg,preferably 70 to 200 mg/kg, preferably 80 to 150 mg/kg, preferably 90 to100 mg/kg.

Compounds of the disclosure may be useful for sensitizing cells toapoptotic signals. Thus, in some embodiments, the compounds of thedisclosure are co-administered with radiation therapy or a secondtherapeutic agent with cytostatic or antineoplastic activity. Suitablecytostatic chemotherapy compounds include, but are not limited to (i)antimetabolites; (ii) DNA-fragmenting agents, (iii) DNA-crosslinkingagents, (iv) intercalating agents (v) protein synthesis inhibitors, (vi)topoisomerase I poisons, such as camptothecin ortopotecan; (vii)topoisomerase II poisons, (viii) microtubule-directed agents, (ix)kinase inhibitors (x) miscellaneous investigational agents (xi)hormones, (xii) hormone antagonists, and (xii) targeted therapies. It iscontemplated that compounds of the disclosure may be useful incombination with any known agents falling into the above 13 classes aswell as any future agents that are currently in development. Inparticular, it is contemplated that compounds of the disclosure may beuseful in combination with current Standards of Care as well as any thatevolve over the foreseeable future. Specific dosages and dosing regimenswould be based on physicians' evolving knowledge and the general skillin the art.

Examples of second therapeutic agents include, but are not limited to, amitotic inhibitor; an alkylating agent; an antimetabolite; a cell cycleinhibitor; an enzyme; a topoisomerase inhibitor; a biological responsemodifier; an anti-hormone; a tubulin inhibitor; a tyrosine-kinaseinhibitor; an antiangiogenic agent such as MMP-2, MMP-9 and COX-2inhibitor; an anti-androgen; a platinum coordination complex(oxaliplatin, carboplatin); a substituted urea such as hydroxyurea; amethylhydrazine derivative; an adrenocortical suppressant, e.g.,mitotane, aminoglutethimide; a hormone and/or hormone antagonist such asthe adrenocorticosteriods (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate), an estrogen (e.g., diethylstilbestrol);an antiestrogen such as tamoxifen; androgen, e.g., testosteronepropionate; and an aromatase inhibitor, such as anastrozole, andAROMASIN (exemestane); a thymidylate synthase inhibitor; a thymidinephosphorylase (TPase) inhibitor; a DNA synthesis inhibitor; and/or atargeted therapy. Exemplary second therapeutic agents include, but arenot limited to, tubulin binding agents including paclitaxel, epothilone,docetaxel, discodermolide, etoposide, vinblastine, vincristine,teniposide, vinorelbine, and vindesine; tyrosine-kinase inhibitorsincluding imatinib, nilotinib, dasatinib, bosutinib, ponatinib, andbafetinib; alkylating antineoplastic agents including busulfan,carmustine, chlorambucil, cyclophosphamide, cyclophosphamide,dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan,mercaptopurine, procarbazine; antimetabolites including cladribine,cytarabine, fludarabine, gemcitabine, pentostatin, 5-fluorouracil,clofarabine, capecitabine, methotrexate, thioguanine; cytotoxicantibiotics including daunorubicin, doxorubicin, idarubicin, mitomycin,actinomycin, epirubicin; topoisomerase inhibitors including irinotecan,mitoxantrone, topotecan; thymidine phosphorylase (TPase) inhibitors suchas tipiracil and trifluridine; DNA synthesis inhibitors such asoxaliplatin; targeted therapies such as cetuximab, bevacizumab,panitumumab, zivaflibercept, ramucirumab; and mixtures thereof.

The combination therapy is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single dosage form having afixed ratio of each therapeutic agent or in multiple, single dosageforms for each of the therapeutic agents. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. Combination therapy also can embrace theadministration of the therapeutic agents as described above in furthercombination with other biologically active ingredients and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises a non-drug treatment, the non-drug treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andnon-drug treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

A treatment method may comprise administering the compound of formula(I) or a pharmaceutical composition containing the compound of formula(I) of the current disclosure in any of its embodiments as a single doseor multiple individual divided doses. In some embodiments, thecomposition is administered at various dosages (e.g., a first dose withan effective amount of 200 mg/kg and a second dose with an effectiveamount of 50 mg/kg). In some embodiments, the interval of time betweenthe administration of the pharmaceutical composition and theadministration of one or more second therapies may be about 1 to 5minutes, 1 to 30 minutes, 30 minutes to 60 minutes, 1 hour, 1 to 2hours, 2 to 6 hours, 2 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 days,3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks,20 weeks, 26 weeks, 52 weeks, 11 to 15 weeks, 15 to 20 weeks, 20 to 30weeks, 30 to 40 weeks, 40 to 50 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 1 year, 2 years, or any period of time in between.Preferably, the composition is administered once daily for at least 2days, at least 5 days, at least 6 days, or at least 7 days. In someembodiments, the pharmaceutical composition and optionally one or moresecond therapies are administered less than 1 day, less than 1 week,less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 1month, less than 2 months, less than 3 months, less than 6 months, lessthan 1 year, less than 2 years, or less than 5 years apart.

The methods for treating cancer and other proliferative disordersdescribed herein inhibit, remove, eradicate, reduce, regress, diminish,arrest or stabilize a cancerous tumor, including at least one of thetumor growth, tumor cell viability, tumor cell division andproliferation, tumor metabolism, blood flow to the tumor and metastasisof the tumor. In some embodiments, the size of a tumor, whether byvolume, weight or diameter, is reduced after the treatment by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100%, relative to the tumor size before treatment. In someembodiments, the size of a tumor after treatment is not reduced but ismaintained at the same size as before treatment. Methods of assessingtumor size include, but are not limited to, CT scan, MRI, DCE-MRI andPET scan.

The method may further comprise measuring a concentration of a biomarkerand/or detecting a mutation in a biomarker before and/or after thepharmaceutical composition comprising the compound of formula (I) of thepresent disclosure is administered. Generic cancer biomarkers includecirculating tumor DNA (ctDNA) and circulating tumor cells (CTC).Exemplary biomarkers for colon cancer include, without limitation,carcinoembryonic antigen (CEA), carbohydrate antigen 242 (CA 242), CA195, CA 19-9, MSI, and 18qLOH. Exemplary biomarkers for breast cancerinclude, without limitation, BRCA1, BRCA2, HER-2, estrogen receptor,progesterone receptor, cancer antigen 15-3, cancer antigen 27.29,carcinoembryonic antigen, Ki67, cyclin D1, cyclin E, and ERβ. Exemplarybiomarkers for stomach cancer include, without limitation,carcinoembryonic antigen (CEA), CA19-9, carbohydrate antigen (CA) 72-4,alpha-fetoprotein, carbohydrate antigen (CA)12-5, SLE, BCA-225, hCG, andpepsinogenI/II.

Potentially predictive cancer biomarkers include, without limitation,mutations in genes BRCA1 and BRCA2 for breast cancer, overexpressions ofTYMS, mutations in genes p53 and KRAS for colon cancer, and highconcentration levels of AFP, and overexpressions of HSP90a for livercancer.

The mutation in the biomarker may be detected by procedures such asrestriction fragment length polymorphism (RFLP), polymerase chainreaction (PCR) assay, multiplex ligation-dependent probe amplification(MLPA), denaturing gradient gel electrophoresis (DGGE), single-strandconformation polymorphism (SSCP), hetero-duplex analysis, proteintruncation test (PTT), and oligonucleotide ligation assay (OLA). Theprocedures to detect the mutation are well-known to those of ordinaryskill in the art.

The concentration level of the cancer biomarker in a sample (i.e.,biological sample obtained from the subject in need of therapy includinga single cell, multiple cells, fragments of cells, a tissue sample,and/or body fluid, for example red blood cells, white blood cells,platelets, hepatocytes, epithelial cells, endothelial cells, a skinbiopsy, a mucosa biopsy, an aliquot of urine, saliva, whole blood,serum, plasma, lymph) may be measured for example by an immunoassay.Typical immunoassay methods include, without limitation, enzyme-linkedimmunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT),Western blotting, immunohistochemistry (IHC), immunocytochemistry,immunostaining, and multiple reaction monitoring (MRM) based massspectrometric immunoassay. The protocol for measuring the concentrationof the biomarker and/or detecting the mutation in the biomarker is knownto those of ordinary skill, for example by performing the steps outlinedin the commercially available assay kit sold by Sigma-Aldrich, ThermoFisher Scientific, R & D Systems, ZeptoMetrix Inc., Cayman Inc., Abcam,Trevigen, Dojindo Molecular Technologies, Biovision, and Enzo LifeSciences.

In some embodiments, a concentration of the biomarker is measured beforeand after the administration. When the concentration of the biomarker ismaintained, the method may further comprise increasing the effectiveamount of the compound of formula (I) by at least 5%, at least 10%, orat least 30%, and up to 80%, up to 60%, or up to 50% of an initialeffective amount. The subject may be administered with the increaseddosage for a longer period (e.g., 1 week more, 2 weeks more, or 2 monthsmore) than the duration prescribed with the initial effective amount.

In some embodiments, the administration is stopped once the subject istreated.

The examples below are intended to further illustrate protocols forpreparing, characterizing, and using the compounds of the presentdisclosure, and are not intended to limit the scope of the claims.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”,“contain(s)”, and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The present disclosurealso contemplates other embodiments “comprising”, “consisting of” and“consisting essentially of”, the embodiments or elements presentedherein, whether explicitly set forth or not.

As used herein, the words “a” and “an” and the like carry the meaning of“one or more.”

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

All patents and other references mentioned above are incorporated infull herein by this reference, the same as if set forth at length.

EXAMPLES Results Chemical Synthesis of3-aryl-2-[(3-arylpropenoyl)amino]-N-substituted-propenamide derivatives(MOS-1503 to MOS-5112)

The synthetic pathway, described in FIG. 1C, utilized Erlenmeyerchemistry for azlactone synthesis. See El-Araby, M. E.; Omar, A. M.;Khayat, M. T.; Assiri, H. A.; Al-Abd, A. M., Molecular Mimics of ClassicP-Glycoprotein Inhibitors as Multidrug Resistance Suppressors and TheirSynergistic Effect on Paclitaxel. PLoS One 2017, 12 (1), e0168938,incorporated herein by reference in its entirety. In this scheme,non-commercially available cinnamic acid analogues 2 were prepared bycondensation of the corresponding aldehyde (1, R₁CHO) with malonic acid.See Zhang, P.; Hu, H.-R.; Bian, S.-H.; Huang, Z.-H.; Chu, Y.; Ye, D.-Y.,Design, synthesis and biological evaluation of benzothiazepinones (BTZs)as novel non-ATP competitive inhibitors of glycogen synthase kinase-3β(GSK-3β). European journal of medicinal chemistry 2013, 61, 95-103,incorporated herein by reference in its entirety. After conversion tothe cinnamoylglycine analogs (3), cyclocondensation was affected bytheir reaction with the corresponding aldehydes (R₂CHO) to afford the2-arylvinyl-4-benzylidene-5-oxazolinone derivatives (azlactones, 4)under Erlenmeyer conditions. The key azlactone intermediate 4 wassubsequently reacted with a variety of aliphatic and aromatic amines tofurnish the final compounds (FIG. 2 and Table 1). Generally, thealiphatic and benzylic amines reacted with the azlactone smoothly atroom temperature in ethanol. The less reactive aromatic amines neededmicrowave heating in the presence of N,N-dimethylformamide (DMF) as asolvent.

Biological Screening

1. Antiproliferative Activities of Bis-(3-arylpropenamide)derivatives

Compounds in this study were tested against colorectal cancer cell lineHCT-116, using sulforhodamine-B (SRB) assay. See Skehan, P.; Storeng,R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.;Bokesch, H.; Kenney, S.; Boyd, M. R., New colorimetric cytotoxicityassay for anticancer-drug screening. J Natl Cancer Inst 1990, 82 (13),1107-12, incorporated herein by reference in its entirety. The subset ofcompounds (Codes MOS-1503 to 1559), having fixed phenyl groups at R₁ andR₂ positions (FIG. 2), demonstrated moderate to weak antiproliferativeactivities (Table 1). However, it was clear that compounds bearinghydrophobic aliphatic groups such as MOS-1505 and MOS-1512 are clearlyfavored in providing anticancer activities. Compounds having non-polargroups on R₃ position were more potent than those having polar groupssuch as MOS-1511, MOS-1532, MOS-1536. The N-aryl substituents were lessadvantageous in both potency and solubility. To zoom on N-aliphaticcompounds, we observed that MOS-1505 N-(1-adamantyl) in this subset wasthe analogue with IC₅₀=14.7±0.3 μM against HCT116 colorectal cancer(CRC) cell lines. Unfortunately, MOS-1505 had poor solubility (ClogP6.08). Meanwhile, compound 1512 was slightly less active (IC₅₀=32.0±2.6μM) but showed a better solubility profile (CLogP 4.48).

TABLE 1 Cytotoxic activities of final compounds on colon cancer celllines Code R¹ R² R³ HCT-116* CACO-2* HT-29* 1503 Ph Ph NH-cyclopentyl48.5 ± 3.1  ND ND 1505 Ph Ph NH-(1-adamantyl) 14.7 ± 0.3  ND ND 1507 PhPh NH-(3-pyridyl) >100 ND ND 1508 Ph Ph NH₂ >100 ND** ND 1511 Ph PhNH-(4-methyl-1- >100 ND ND piperazinyl) 1512 Ph Ph NH-(n-Pr) 32.0 ± 2.638.0 ± 0.87 35.5 ± 1.7  1513 Ph Ph NH-(3-CN-Ph) >100 ND ND 1528 Ph PhNH-(4-F-Ph) 91.9 ± 4.4  ND ND 1530 Ph Ph NH-furfuryl >100 ND ND 1531 PhPh NH-(2- >100 ND ND morpholinoethyl) 1532 Ph PhNH-(2-hydroxyethyl) >100 ND ND 1536 Ph Ph 4-morpholinyl >100 ND ND 1555Ph Ph 1-pyrrolidinyl >100 ND ND 1556 Ph Ph NH-(n-Bu) 67.2 ± 6.3  ND ND1557 Ph Ph NH-(sec-Bu) 52.4 ± 7.1  ND ND 1558 Ph Ph NH-Et >100 ND ND1559 Ph Ph N(Me)Et 28.8 ± 1.0  ND ND 1812 4ClPh Ph NH-(n-Pr) 10.3 ± 0.3410.7 ± 0.5  7.90 ± 0.43 1912 4-MeOPh Ph NH-(n-Pr) 34.1 ± 1.8  18.5 ±1.5  55.3 ± 3.2  2012 4-MePh Ph NH-(n-Pr) 35.2 ± 0.54 12.7 ± 0.67 54.1 ±0.42 2112 2-thienyl Ph NH-(n-Pr) 43.0 ± 1.5  27.8 ± 1.3  43.2 ± 2.3 2212 Ph 4-Ph NH-(n-Pr) 15.2 ± 1.2  13.2 ± 1.0  7.20 ± 0.31 2312 4-ClPh4-FPh NH-(n-Pr) 3.80 ± 0.24  2.60 ± 0.233 4.00 ± 0.14 2412 4-MeOPh 4-FPhNH-(n-Pr) 29.3 ± 1.2  45.73 ± 1.2  86.5 ± 2.9  2512 4-MePh 4-FPhNH-(n-Pr) 6.70 ± 0.36 5.10 ± 0.17 11.80 ± 0.18  2612 2-thienyl 4-FPhNH-(n-Pr) 17.3 ± 0.13 10.7 ± 0.68 7.68 ± 0.05 2712 Ph 4-(Me₂N)PhNH-(n-Pr) 6.95 ± 0.46 4.24 ± 0.09  6.8 ± 0.05 2812 4-ClPh 4-(Me₂N)PhNH-(n-Pr) 20.7 ± 0.32 17.0 ± 0.8  9.52 ± 0.42 2912 4-MeOPh 4-(Me₂N)PhNH-(n-Pr) 12.4 ± 0.25 15.3 ± 0.05 36.9 ± 0.25 3012 4-MePh 4-(Me₂N)PhNH-(n-Pr) 22.1 ± 0.10 29.4 ± 0.08 39.6 ± 0.22 3112 2-thienyl 4-(Me₂N)PhNH-(n-Pr) 11.6 ± 0.39 27.7 ± 3.7  66.5 ± 0.10 3212 Ph 3-MeO—4-OHPhNH-(n-Pr) >100 6.32 ± 0.02 9.14 ± 0.23 3312 4-ClPh 3-MeO—4-OHPhNH-(n-Pr) 37.8 ± 1.8   6.1 ± 0.44  5.4 ± 0.34 3412 4-MeOPh 3-MeO—4-OHPhNH-(n-Pr) 38.4 ± 2.1  18.0 ± 1.2  48.6 ± 0.4  3512 4-MePh 3-MeO—4-OHPhNH-(n-Pr) 26.3 ± 1.3  20.9 ± 1.1  15.1 ± 0.67 3612 2-thienyl3-MeO—4-OHPh NH-(n-Pr) 24.8 ± 1.4  28.3 ± 1.3  48.3 ± 1.9  3712 Ph3-indolyl NH-(n-Pr) 1.65 ± 0.05  2.1 ± 0.03 2.36 ± 0.04 3812 4-ClPh3-indolyl NH-(n-Pr) 3.51 ± 0.21 3.35 ± 0.05 3.41 ± 0.03 3912 4-MeOPh3-indolyl NH-(n-Pr) 4.35 ± 0.29 2.07 ± 0.03 2.49 ± 0.04 4012 4-MePh3-indolyl NH-(n-Pr) 3.39 ± 0.10  2.3 ± 0.08 3.12 ± 0.06 4112 2-thienyl3-indolyl NH-(n-Pr) 2.85 ± 1.5  0.89 ± 0.04 1.65 ± 0.07 4212 Ph 3-yridylNH-(n-Pr) 73.0 ± 2.5  66.0 ± 2.2  >100 4312 4-ClPh 3-pyridyl NH-(n-Pr)49.4 ± 1.9  47.3 ± 1.9  36.0 ± 1.7  4412 4-MeOPh 3-pyridyl NH-(n-Pr)52.1 ± 1.6  38.1 ± 2.0  72.7 ± 6.4  4512 4-MePh 3-pyridyl NH-(n-Pr) >10027.0 ± 1.8  >100 4612 2-thienyl 3-pyridyl NH-(n-Pr) >100 41.8 ± 1.4 68.8 ± 6.9  4712 Ph 4-NO₂Ph NH-(n-Pr) 17.3 ± 1.3  44.2 ± 2.7  23.2 ±1.9  4812 4-ClPh 4-NO₂Ph NH-(n-Pr) >100 >100 36.9 ± 1.5  4912 4-MeOPh4-NO₂Ph NH-(n-Pr) >100 >100 74.3 ± 3.6 5012 4-MePh 4-NO₂PhNH-(n-Pr) >100 >100 >100 5112 2-thienyl 4-NO₂Ph NH-(n-Pr) >100 34.2 ±1.1  >100 tCA 12.4 ± 1.2  ND ND Doxo  0.6 ± 0.10 0.14 ± .01   0.3 ± 0.01*IC₅₀ values in μM ± Standard Error of Means (SEM). **ND, NotDetermined. tCA - trans-cinnamaldehyde Doxo - doxorubicin

MOS-1512 showed significantly low cancer cell resistance (R-value=8.2%),and high selectivity in killing cancer cells (HCT-116, Caco-2 and HT-29)versus highly proliferative normal cells (C-166 mouse skin fibroblasts)(FIG. 3A). See El-Araby, M. E.; Omar, A. M.; Khayat, M. T.; Assiri, H.A.; Al-Abd, A. M., Molecular Mimics of Classic P-Glycoprotein Inhibitorsas Multidrug Resistance Suppressors and Their Synergistic Effect onPaclitaxel. PLoS One 2017, 12 (1), e0168938, incorporated herein byreference in its entirety.

Based on the favorable pharmacologic and physicochemical properties ofMOS-1512, it was selected as a new lead for the second subset ofcompounds (MOS-1812 to MOS-5112). The compounds in this series containedvariable groups at R¹ and R² that were all tested against the threecancer cell lines (HCT-116, Caco-2 and HT-29). Some of the compoundsshowed high potencies similar to the reference drug doxorubicin (Table1). For instance, MOS-2312, MOS-2812, MOS-3712, MOS-3812, MOS-3912,MOS-4012 and MOS-4112 demonstrated excellent cytotoxic activitiesagainst CRC cell lines (under 5 μM). For instance, compound MOS-4112killed cancer cells HCT-116, Caco-2, and HT-29 cells in high potencies(IC₅₀ of 2.85±1.5, 0.89±0.04 and 1.65±0.07 μM, respectively) that arecomparable to doxorubicin (0.6±0.1 0.14±0.05, and 0.3±0.01 μM,respectively). Compound MOS-4112 also had desirable selectivity againstcancer cells versus baby hamster kidney (BHK) cell lines (IC₅₀=77.6±6.2μM)(FIG. 3B). Also, potent compound 3712 is noteworthy, which exhibitedIC₅₀ of 1.65±0.05, 2.1±0.03 μM and 2.36±0.04 against HCT-116, Caco-2,and HT-29 cells, respectively.

2. Induction of Apoptosis in HCT-116 Cancer Cell Lines Upon Treatmentwith MOS-1512 and MOS-4112

Several assays were performed to determine the apoptotic effect of theearly lead MOS-1512 as well as the potent derivative MOS-4112 on HCT-116cells. First, the effect of 1512 on morphological changes of HCT-116 wasmonitored by staining the cell with the dye4′,6′-diamidino-2-phenylindole (DAPI) after 48 hours of treatment with32 μM of 1512 (IC₅₀ concentration). A very strong blue fluorescence wasobserved, indicative of increased cell membrane permeability and uptakeof the dye, indicating that 1512 is able to disrupt the integrity of thecell to induce apoptosis (FIG. 4A). See Drutovic, D.; Chripkova, M.;Pilatova, M.; Kruzliak, P.; Perjesi, P.; Sarissky, M.; Lupi, M.; Damia,G.; Broggini, M.; Mojzis, J., Benzylidenetetralones, cyclic chalconeanalogues, induce cell cycle arrest and apoptosis in HCT116 colorectalcancer cells. Tumour Biol 2014, 35 (10), 9967-75, incorporated herein byreference in its entirety.

DAPI staining test was also performed after incubation of HCT-116 cellswith 4112 at 2.85 μM (the IC₅₀ value), and the results at 24 and 48 hshown in FIG. 4B. The percentage of cells with fragmented DNA andcondensed nuclei greatly increased after 48 h compared to 24 h.

3. Cell Cycle Distribution Upon Treatment with MOS-4112

The effect of 4112 on HCT-116 cell cycle distribution was examined atthe respective IC₅₀ concentrations for 24 h using a FACS Calibur flowcytometer. The exposure of the cells to 4112 led to a significantincrease in the proportion of cells in pre-G1 phase (Up to 9-foldcompared to the control). Accumulation of cells in pre-G1 phase, likelyas a result of degradation or fragmentation of genetic materialindicates a possible role for apoptosis through compound-induced growthinhibition.

In the subsequent similar experiment, 4112-treated HCT-116 cells weremonitored for apoptotic changes at 24 h and 48 h. Cell cycle populationsfor the treated cells were determined by flow cytometry analysis afterstaining with Annexin V/Propidium iodide (FIG. 5) and revealed a largetime-dependent increase in the percentage of HCT-116 apoptotic cells. At24 hours, there was very little change in cell viability, while after 48hours, viable cells have decreased to 72% (down from 99.9%). In thecontext, cell populations in both early and late apoptosis increasedfrom almost zero to 17.5% and 4.1%, respectively.

4. Elevation of Oxidative Stress Indicators within HCT-116 Cells UponTreatment with MOS-4112.

The elevation of oxidative stress inside cancer cells, consequent totreatment with 4112, was investigated via monitoring ROS release overtime (24 and 48 h). In this test, the 2′,7′-dichlorofluorescin diacetate(DCFDA) dye was used to detect various ROS species and emit greenfluorescence. It was clear that compound MOS-4112 causes accumulation ofROS, compared to control (untreated cells) and it is proportional to thetime of exposure (FIG. 6).

5. Inhibition of Cancer Stem Cell Proliferation in HT-29 Cancer CellLines Upon Treatment with MOS-3712 and MOS-4112

Compound 4112 and another potent compound 3712 were also evaluated fortheir ability to inhibit cancer stem cell proliferation using aprimary(1°) colonosphere formation assay. In this assay, HT-29 coloncancer cells were grown in low adhesion plates to form spheres aspreviously described. See Patel, N. J.; Karuturi, R.; Al-Horani, R. A.;Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B., Synthetic,non-saccharide, glycosaminoglycan mimetics selectively target coloncancer stem cells. ACS Chem Biol 2014, 9 (8), 1826-33, incorporatedherein by reference in its entirety. Wells treated with the inventivecompounds were compared to vehicle treated cells for primary spheregrowth inhibition after 5 days of incubation. Results, as illustrated inFIG. 7, showed a dose-dependent inhibition of colon cancer spheroidsformation (>50 μm). The IC₅₀ of the two compounds were 21.12±1.3 μM and18.85±1.1 μM for 3712 and 4112 respectively, reflecting similarcloseness in potency as in HT-29 monolayer cell line assay (2.36±0.04 μMand 1.65±0.07 μM, respectively). Of note, monolayer growth conditionsare ideally suited for examining cellular proliferation, whereasspheroid growth conditions examine cancer stem cell growth andself-renewal properties. Hence, differential potency of the molecules inthe two condition is reflective of their effects on two differentphenotypes.

6. Lethal Dose Toxicity Test with Compounds MOS-1512, MOS-3712 andMOS-4112

Since compounds with electrophilic properties, e.g., MA are usuallyinvolved in a variety of toxic effects on mammalians, the general acutetoxicity of 1512, 3712 and 4112 was investigated by determining the invivo lethal dose (LD₅₀) according to the Globally HarmonizedClassification System (GHS) and following the Organization for EconomicCooperation and Development (OECD) guideline 423 (modified, adopted Mar.23, 2006). As observed in the cellular assay, the compound safety wasconfirmed, as the LD₅₀ for all compounds were between 2000-5000 mg/kg(Category 5). See Spielmann, H.; Genschow, E.; Liebsch, M.; Halle, W.,Determination of the starting dose for acute oral toxicity (LD50)testing in the up and down procedure (UDP) from cytotoxicity data. ATLA1999, 27 (6), 957-966, incorporated herein by reference in its entirety.

Brief Summary of Results

The compounds showed selective toxicity against cancer cell lines (inparticular colorectal cancer cells) and cancer stem cells (CSC) overnoncancerous cells (C-166 and BHK), low animal toxicity, and a clearcell death mechanism. The two most potent compounds, 3712 and 4112exhibited significant anti-proliferative effects on colorectal cancercell lines CaCo-2, HCT-116 and HT-29.

Experimental

All melting points were uncorrected and measured using the capillarymelting point instrument BI 9100 (Barnstead Electrothermal, UK).Infrared spectra were recorded on a Thermo Scientific Niccolet iS10FT-IR Spectrometer (King Fand Center for Medical Research, KingAbdulaziz University, Jeddah, Saudi Arabia). Only the important IRstretching bands were listed, including NH, OH, CH, C═O, C═N and/or C═C.In FT-IR, all samples were measured neat. ¹H NMR spectra were determinedon an AVANCE-III 600 MHz and AVANCE-III HD 850 MHz spectrometers(Bruker, Germany), and chemical shifts are expressed as ppm against TMSas an internal reference (King Fand Center for Medical Research andFaculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia).LC/MS analyses were performed on an Agilent 6320 Ion TrapHPLC-ESI-MS/DAD (Santa Clara, Calif., USA) with the following settings:The analytes were separated using an Macherey-Nagel Nucleodur-C18 column(150 mm length×4.6 mm i.d., 5 μm) (Macherey-Nagel GMBH & Co. KG, Duren,Germany). Mobile phase; isocratic elution using a mixture of isopropanoland 0.01M ammonium acetate in water (65:35, v/v). The flow rate was 0.4mL/min; total run time=20 min. Purities are reported according topercentage of Peak Areas at wavelength 280 nm. High-resolution massspectrometry (HRMS) was performed in the Faculty of Science, KingAbdulaziz University on Impact II™ Q-TOF spectrometer (Bruker, Germany).Microanalyses were operated using Vario, an Elmentar apparatus(Shimadzu, Japan), Organic Microanalysis Unit, Cairo University, Giza,Egypt. Column chromatography was performed on a silica gel 60 (particlesize 0.06 mm-0.20 mm).

Chemical Synthesis

The previously reported intermediate azlactone 4a (see FIG. 1C,intermediate 4, where R¹ and R² are phenyl, i.e.,4-((Z)-benzylidene)-2-((E)-styryl)oxazol-5(4H)-one) (Fahmy, A.; Orabi,M., Reactions of 4-arylidene-2-styryl-5(4)-oxazolones and relatedcompounds, Indian Journal of Chemistry 1972, 10 (10), 961-964), wasprepared according to procedures mentioned below, and their physical andspectral properties were confirmed. Purity of compounds were firstassessed qualitatively using Thin Layer Chromatography (TLC), ¹H NMR andquantitatively using LC/MS (UV detection). The compounds were screenedonly if purity was confirmed to be above 95%. The compounds subjected toall biological screenings were used as a single Z isomer as detected byTLC, LC/MS and NMR.

(Z)-2-cinnamamido-N-cyclopentyl-3-phenylacrylamide (1503)

Azlactone 4a (10 mmol, 2.773 g) was dissolved in 50 mL ethanol,cyclopentylamine (20 mmol, 2.0 mL) was then added, and the mixture wasstirred for 2 h. The solvent was removed under reduced pressure. Theresidue was treated with ice-cooled dilute HCl resulting inprecipitation of a off-white solid product. The crude product waspurified by crystallization from ethanol. The purified product 1503 wasan off-white solid, (0.45 g, 60%), Mp 239° C. ¹H NMR (600 MHz,acetone-d₆) δ_(H) ppm 8.85 (s, 1H), 7.59-7.67 (m, 2H), 7.55 (d, J=7.53Hz, 2H), 7.40-7.47 (m, 3H), 7.37 (t, J=7.53 Hz, 2H), 7.28-7.32 (m, 1H),7.09 (br. s., 1H), 6.96 (d, J=15.81 Hz, 1H), 4.24-4.32 (m, 1H),1.90-1.98 (m, 2H), 1.71 (br. s., 2H), 1.54-1.62 (m, 4H).

(Z)—N-(1-adamantanyl)-2-cinnamamido-3-phenylacrylamide (1505)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) and1-aminoadamantane (2 mmol, 0.308 g). The product 1505 was a white solid(0.75 g, 88%), Mp 272° C. ¹H NMR (600 MHz, acetone-d₆) δ_(H) ppm 8.86(s, 1H), 7.62-7.68 (m, 3H), 7.54 (d, J=7.53 Hz, 2H), 7.40-7.47 (m, 3H),7.37 (t, J=7.53 Hz, 2H), 7.27-7.32 (m, 1H), 7.05 (s, 1H), 6.98 (d,J=15.43 Hz, 1 H), 2.17 (m, 1H), 2.13 (br. s., 6H), 2.00-2.03 (m, 1H),1.95-1.97 (m, 1H), 1.73 (br. s., 6H), 1.57-1.59 (m, 1H).

(Z)-2-[(E)-cinnamamido]-3-phenyl-N-(3-pyridyl)acrylamide (1507)

In the reaction tube of a microwave reactor (SynthLab), azlactone 4a (2mmol, 0.554 g) and 3-aminopyridine (2 mmol, 0.188 g) were mixed with 5mL DMF. The reaction was heated to 200° C. while stirring in a Milestonemicrowave reactor for 10 min. After cooling, the mixture was addedslowly to ice-cold water. The resulting solid was collected byfiltration, washed with water and purified using silica gelchromatography (petroleum ether/dichloromethane (DCM)/MeOH, gradient) togive 1507 as a white solid (0.206 g, 30%), Mp>250° C. (dec). ¹H NMR (600MHz, Acetone-d₆) δ_(H) ppm 9.79 (1H, br s), 9.31 (1H, br s), 8.91 (1H,s), 8.31 (1H, dd, J=6.52, 1.13 Hz), 8.27 (1H, d, J=8.66 Hz), 7.69-7.61(5H, m), 7.48-7.39 (5H, m), 7.38-7.32 (2H, m), 7.21 (1H, s), 7.03 (1H,d, J=15.81 Hz), LC-MS (ESI), RT=7.4 min, m/z 370.2 [M+H]⁺.

N—[(Z)-3-amino-3-oxo-1-phenylprop-1-en-2-yl]-(E)-cinnamamide (1508)

Azlactone 4a (2 mmol, 0.554 g) was stirred in 5 mL solution of ammonia(2M) in ethanol for 2 h. The product was precipitated as white powderwhich was filtered, washed with water several times followed by ethanoland dichloromethane. Crystallization from aqueous methanol provided awhite solid of 1508 Mp>250° C. (dec.) ¹H NMR (DMSO-d6) d_(H) ppm 10.11(br. s., 1H), 7.75 (br. s., 1H), 7.61 (d, J=6.78 Hz, 2H), 7.55 (d,J=7.53 Hz, 2H), 7.35-7.53 (m, 6H), 7.31 (d, J=7.15 Hz, 1H), 7.16 (br.s., 1H), 7.10 (s, 1H), 6.91 (d, J=15.81 Hz, 1H).

(Z)-2-[(E)-cinnamamido]N-(4-methyl-1-piperazinyl)-3-phenyl-acrylamide(1511)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) and1-amino-4-methylpiperazine (4 mmol, 0.49 mL). The product 1511 was anoff-white power, (305 mg, 39%), Mp. 190° C.; 1H NMR (600 MHz, DMSO-d6)δ_(H) ppm 9.83 (s, 1H), 9.58 (s, 1H), 7.62 (d, J=7.53 Hz, 2H), 7.54-7.58(m, J=7.91 Hz, 2H), 7.52 (d, J=15.81 Hz, 1H), 7.44-7.48 (m, 2H),7.39-7.44 (m, 3H), 7.30-7.35 (m, 1H), 6.91 (d, J=15.81 Hz, 1H), 6.75(br. s., 1H), 3.43 (br. s., 2H), 3.14-3.22 (m, 4H), 3.10 (br. s., 2H),2.77 (br. s., 3H).

(Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide (1512)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andn-propylamine (2 mmol, 0.166 mL). The product 1512 was a white solid(542 mg, 81%), Mp 207° C. ¹H NMR (850 MHz, DMSO-d₆) δ_(H) 9.69 (s, 1H),8.12 (t, J=5.97 Hz, 1H), 7.62 (d, J=7.78 Hz, 2H), 7.55 (d, J=7.78 Hz,2H), 7.50 (d, J=16.09 Hz, 1H), 7.46 (t, J=7.79 Hz, 2H), 7.42 (t, J=7.79Hz, 1H), 7.38 (t, J=7.79 Hz, 2H), 7.31 (t, J=7.79 Hz, 1H), 7.00 (s, 1H),6.87 (d, J=16.09 Hz, 1H), 3.12 (q, J=6.75 Hz, 2H), 1.49 (dt, J=7.27 and6.9 Hz, 2H), 0.88 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ165.1, 164.7, 140.0, 134.8, 134.3, 130.5, 129.9, 129.4, 129.1, 128.6,128.6, 127.8, 126.9, 121.6, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3,39.2, 22.3, 11.5; LC-MS (ESI), RT=3.6 min, m/z 335.1 [M+H]⁺. Anal. Calcdfor (C₂₁H₂₂N₂O₂): C, 75.42; H, 6.63; N, 8.38 Found C, 75.37; H, 6.59; N,8.29.

(Z)-2-[(E)-cinnamamido]N-(3-cyanophenyl)-3-phenyl-acrylamide (1513)

This compound was prepared according to the procedure used in thesynthesis of compound 1503 starting from the azlactone 4a (2 mmol, 0.554g) and 3-aminobenzonitrile (2 mmol, 0.236 g). The product 1513 as awhite solid (0.352 g, 47%), Mp 235-237° C. ¹H NMR (600 MHz, DMSO-d₆)δ_(H) 10.52 (1H, br s), 10.05 (1H, br s), 8.19 (s, 1H), 8.01 (1H, d,J=7.78 Hz), 7.66-7.60 (m, 4H), 7.58-7.50 (m, 3H), 7.48-7.40 (m, 5H),7.36 (m, 1H), 6.98 (s, 1H), 6.93 (1H, d, J=16.09 Hz).

(Z)-2-[(E)-cinnamamido]N-(4-fluorophenyl)-3-phenylacrylamide (1528)

This compound was prepared according to the procedure used in thesynthesis of compound 1503 starting from the azlactone 4a (2 mmol, 0.554g) and 4-fluoroaniline (2 mmol, 0.192 mL). The final compound 1528 wascollected as a white solid (0.471 g, 65%), Mp 200° C. ¹H NMR (600 MHz,DMSO-d₆) δ_(H) ppm 10.18 (1H, s), 9.92 (1H, s), 7.74-7.68 (2H, m), 7.62(4H, t, J=6.96 Hz), 7.51 (1H, d, J=15.81 Hz), 7.48-7.39 (7H, m), 7.35(1H, m,), 7.17 (2H, m), 6.96 (s, 1H), 6.91 (1H, d, J=15.81 Hz); IR(FT-IR, cm⁻¹): 3214.5, 3059.3, 2979.2, 1647.5, 1614.2, 1505.4.

(Z)-2-[(E)-cinnamamido]N-(2-furylmethyl)-3-phenyl-acrylamide (1530)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andfurfurylamine (2 mmol, 0.176 mL). The product 1530 was an off-whitesolid, (0.45 g, 60%), Mp 198-199° C. ¹H NMR (600 MHz, DMSO-d₆) δ_(H)9.76 (1H, s), 8.62 (1H, t, J=5.8 Hz), 7.63 (2H, m), 7.55-7.60 (3H, m),7.53 (1H, d, J=15.8 Hz), 7.46 (2H, m), 7.41 (1H, d, J=7.5 Hz), & 0.39(2H, t, J=7.7 Hz), 7.32 (1H, m), 7.10 (1H, s), 6.88 (1H, d, J=15.8 Hz),6.41 (1H, m), 6.32 (1H, m), 4.37 (2H, d, J=5.65 Hz); IR (FT-IR, cm⁻¹):3394.3, 3065.2, 2955.9, 1705.5, 1625.7, 1508.2; LC-MS (ESI) RT=3.6 min,m/z 373.1[M+H]⁺. Anal. Calcd for (C₂₃H₂₀N₂O₃): C, 74.18; H, 5.41; N,7.52; Found C, 73.79; H, 5.08; N, 7.65.

(Z)-2-[(E)-cinnamamido]-N-(2-morpholinoethyl)-3-phenyl-acrylamide (1531)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) and2-(4-morpholinyl)ethylamine (5 mmol, 0.65 mL). The product 1531 was anoff-white solid, (0.61 g, 75%), Mp 169° C. ¹H NMR (600 MHz, DMSO-d₆)δ_(H) 9.74 (s, 1H), 7.96 (br s, 1H), 7.63 (d, J=7.15 Hz, 2H), 7.56 (d,J=7.53 Hz, 2H), 7.53 (d, J=15.81 Hz, 1H), 7.37-7.50 (m, 5H), 7.29-7.36(m, 1H), 7.07 (s, 1H), 6.88 (d, J=16.19 Hz, 1H), 3.52-3.61 (m, 4H),3.25-3.32 (m, 2H), 2.37-2.47 (m, 6H). LC-MS (ESI) RT=3.7 min, m/z 406.2[M+H]+.

(Z)-2-[(E)-cinnamamido]-N-(2-hydroxyethyl)-3-phenyl-acrylamide (1532)

Ethanolamine (10 mmol, 0.6 m) was placed in a conical flask and stirred,added Azlactone 4a (2 mmol, 0.554 g) portion wise while stirring.Reaction was left to go to completion for two hours. The mixture wastreated with ice-cooled water containing 10 mL 1M HCl and theprecipitated product was collected by filtration. Purification wascarried out by crystallization in ethanol to furnish 1532 as a whitefluffy solid (0.43 g, 63.9%), Mp 204° C., ¹H NMR (850 MHz, DMSO-d₆)δ_(H) 9.72 (s, 1H), 8.06 (t, J=5.71 Hz, 1H), 7.62 (d, J=7.79 Hz, 2H),7.55 (d, J=7.79 Hz, 2H), 7.51 (d, J=15.57 Hz, 1H), 7.49-7.44 (m, 2H),7.42 (d, J=7.27 Hz, 1H), 7.39 (t, J=7.79 Hz, 2H), 7.29-7.34 (m, 1H),6.88 (d, J=16.09 Hz, 1H) 7.05 (s, 1H), 4.65 (t, J=5.71 Hz, 1H), 3.47 (q,J=5.88 Hz, 2H), 3.24 (q, J=6.23 Hz, 2H). LC-MS (ESI) RT=3.48 min, m/z337.0 [M+H]⁺.

N—[(Z)-3-morpholino-3-oxo-1-phenylprop-1-en-2-yl]cinnamamide (1536)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andmorpholine (4 mmol, 0.39 mL). The product 1536 was an off-white power,(0.514 g, 71%), Mp. 185° C.; 1H NMR (600 MHz, DMSO-d6) δ_(H) 10.07 (s,1H), 7.56-7.63 (m, 6H), 7.40-7.48 (m, 6H), 7.27-7.36 (m, 1H), 6.93 (d,J=15.81 Hz, 1H), 6.19 (s, 1H), 3.53 (br. s., 4H), 3.35 (br. s., 4H).

N—[(Z)-3-(1-pyyrrolidinyl)-3-oxo-1-phenylprop-1-en-2-yl]cinnamamide(1555)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andpyrrolidine (2 mmol, 0.165 mL). The product 1555 was a white power,(0.580 g, 84%), Mp. 194° C.; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 10.00 (s,1H), 7.52-7.64 (m, 6H), 7.37-7.49 (m, 6H), 7.30-7.33 (m, 1H), 6.91 (d,J=15.81 Hz, 1H), 6.32 (s, 1H), 3.58 (t, J=6.02 Hz, 2H), 3.37 (t, J=6.59Hz, 2H), 1.82-1.87 (m, 4H).

(Z)—N-(n-butyl)-2-[(E)-cinnamamido]-3-phenylacrylamide (1556)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andn-butylamine (2 mmol, 0.176 mL). The product 1556 was an off-whitesolid, (0.45 g, 60%), Mp 198-199° C. ¹H NMR (600 MHz, DMSO-d₆) δ_(H)9.69 (s, 1H), 8.10 (t, J=5.83 Hz, 1H), 7.62 (d, J=7.15 Hz, 2H), 7.56 (d,J=7.53 Hz, 2H), 7.51 (d, J=15.81 Hz, 1H), 7.44-7.48 (m, 2H), 7.36-7.44(m, 3H), 7.31 (t, J=7.34 Hz, 1H), 7.01 (s, 1H), 6.88 (d, J=16.19 Hz,1H), 3.17 (q, J=6.78 Hz, 2H), 1.47 (quin, J=7.25 Hz, 2H), 1.32 (sxt,J=7.38 Hz, 2H), 0.90 (t, J=7.34 Hz, 3H).

(Z)—N-(sec-butyl)-2-cinnamamido-3-phenylacrylamide (1557)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andsec-butylamine (2 mmol, 0.202 mL). The product 1557 was an off-whitesolid, (0.51 g, 73%), Mp 229° C. ¹H NMR (600 MHz, DMSO-d₆) δ_(H) ppm9.66 (s, 1H), 7.84 (d, J=8.66 Hz, 1H), 7.62 (d, J=7.15 Hz, 2H), 7.56 (d,J=7.91 Hz, 2H), 7.52 (d, J=15.81 Hz, 1H), 7.49-7.41 (m, 3H), 7.39 (t,J=7.72 Hz, 2H), 7.34-7.28 (m, 1H), 6.94-6.86 (m, 2H), 3.81 (dt, J=13.93,7.34 Hz, 1H), 1.39-1.59 (m, 2H), 1.11 (d, J=6.78 Hz, 3H), 0.84-0.93 (m,3H).

(Z)-2-[(E)-cinnamamido]-N-ethyl-3-phenylacrylamide (1558)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andethylamine (1 mL of 40% in water). The product 1558 was an off-whitesolid, (0.39 g, 61%), Mp 190° C. ¹H NMR (600 MHz, DMSO-d₆) δ_(H) ppm1.08 (t, J=7.34 Hz, 3H) 3.19 (dd, J=7.15, 6.02 Hz, 2H) 6.87 (d, J=15.81Hz, 1H) 7.02 (s, 1H) 7.28-7.33 (m, 1H) 7.39 (t, J=7.72 Hz, 2H) 7.43 (d,J=7.15 Hz, 1H) 7.44-7.49 (m, 2H) 7.51 (d, J=16.19 Hz, 1H) 7.55 (d,J=7.53 Hz, 2H) 7.62 (d, J=7.15 Hz, 2H) 8.11-8.17 (m, 1H), 9.68 (s, 1H).

(Z)-2-cinnamamido-N-ethyl-N-methyl-3-phenylacrylamide (1559)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from azlactone 4a (2 mmol, 0.554 g) andN-ethyl-N-Methylamine (2 mmol, 0.171 mL). The product 1559 was a whitesolid (0.61 g, 91%), Mp 169° C. ¹H NMR (600 MHz, DMSO-d₆) δ_(H) ppm10.01 (br. s., 1H), 7.61 (d, J=7.15 Hz, 2H), 7.52-7.59 (m, 3H),7.37-7.49 (m, 5H), 7.27-7.34 (m, 1H), 6.94 (d, J=15.81 Hz, 1H),6.09-6.20 (s, 1H), 3.08 (s, 3H), 2.85 (q, J=7.1 Hz, 2H), 1.12 (t, J=6.9Hz, 3H).

Ethyl (Z)-2-cinnamamido-3-phenylacrylate (15EE)

The azlactone 4a (2 mmol, 0.554 g) was heated under reflux in absoluteethanol in presence of 5 mg of 4-dimethylaminopyridine (DMAP) for 3hours. The solvent was removed by vacuum evaporation and residue waspartitioned between dichloromethane and 1M HCl. The organic layer waswashed with water then brine, dried with sodium sulfated and evaporatedunder vacuum to give the product 15EE (ethyl ester) as an off-whitesolid (0.51 g, 79%), Mp 153° C. ¹H NMR (600 MHz, CDCl₃) δ_(H) 8.97 (br.s., 1H), 7.62-7.69 (m, 5H), 7.34-7.50 (m, 6H), 7.30 (s, 1H), 6.99 (d,J=15.81 Hz, 1H), 4.27 (q, J=7.03 Hz, 2H), 1.31 (t, J=7.15 Hz, 3H).

(Z)—N-(n-propyl)-2-(benzoylamino)-3-phenylacrylamide (1612)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from 4-benzylidene-2-phenyloxazol-5(4H)-one(0.25 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 1612was an off-white solid, (0.27 g, 81%), Mp 169° C. ¹H NMR (850 MHz,DMSO-d₆) δ_(H) 10.17 (br s, 1H), 8.32 (br s, 1H), 8.00 (d, J=7.27 Hz,1H), 7.88-7.86 (m, 1H), 7.61-7.55 (m, 1H), 7.53-7.47 (m, 1H), 7.40 (d,J=8.30 Hz, 1H), 7.20-7.28 (m, 2H), 3.11 (m, J=6.75 Hz, 2H), 1.47 (m,1H), 0.86 (t, J=7.27 Hz, 3H). LC-MS (ESI) RT=4.52 min, m/z 309.0 [M+H]+.

(2Z,4E)-2-cinnamamido-5-phenyl-N-propylpenta-2,4-dienamide (1712)

Compound 1712 (El-Araby, M. E.; Omar, A. M.; Khayat, M. T.; Assiri, H.A.; Al-Abd, A. M., Molecular Mimics of Classic P-Glycoprotein Inhibitorsas Multidrug Resistance Suppressors and Their Synergistic Effect onPaclitaxel. PLoS One 2017, 12 (1), e0168938, incorporated herein byreference in its entirety) was prepared according to the proceduredescribed for the synthesis of 1503 starting from(Z)-4-[(E)-3-(phenylallylidene)-2-(E)-styryl]oxazol-5(4H)-one (0.3 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 1712 was a lightyellow solid, (0.314 g, 87%), Mp 186° C. ¹H NMR (850 MHz, DMSO-d₆) δ_(H)9.69 (s, 1H), 8.02 (t, J=5.71 Hz, 1H), 7.64 (d, J=7.27 Hz, 2H),7.55-7.52 (m, 3H), 7.47-7.45 (m, 2H), 7.42-7.40 (m, 1H), 7.38-7.35 (m,2H), 7.31-7.28 (t, J=7.26 Hz, 1H), 7.05-7.00 (dd, 1H, J=15.8 and 10.7Hz), 6.92-6.87 (m, 2H), 6.73 (d, J=10.90 Hz, 1H), 3.12-3.08 (q, J=8.6Hz, 2H), 1.50-1.44 (m, 2H), 0.94 (t, J=7.53 Hz, 1H), 0.87 (t, J=7.27 Hz,3H), LC-MS (ESI) RT=5.25 min, m/z 361.1 [M+H]+(The compound contained24.2% of E-isomer(2E,4E)-2-cinnamamido-5-phenyl-N-propylpenta-2,4-dienamide according toLC-MS (UV) determination. All data are reported for the Z isomer.

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-3-phenyl-N-propylacrylamide(1812)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-benzylidene]-2-[(E)-4-chlorostyryl]oxazol-5(4H)-one (0.310 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 1812 was a whitesolid (325 mg, 88%), Mp 190-191° C. IR (KBr, ν_(max) cm⁻¹) 3281, 2958,1651, 1611; ¹H NMR (850 MHz, DMSO-d₆) δ_(H) 9.71 (br. s., 1H), 8.12 (t,J=5.71 Hz, 1H), 7.64 (d, J=8.30 Hz, 2H), 7.46-7.58 (m, 4H), 7.38 (t,J=7.79 Hz, 2H), 7.31 (t, J=7.79 Hz, 1H), 7.00 (s, 1H), 6.87 (d, J=16.09Hz, 1H), 3.12 (q, J=6.75 Hz, 2H), 1.49 (sxt, J=7.27 Hz, 2H), 0.88 (t,J=7.27 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 165.1, 164.5, 138.7,134.3, 133.8, 130.4, 129.5, 129.4, 129.2, 128.7, 128.6, 127.0, 122.4,41.1, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 11.5; LC/MS (ESI),RT=4.8 min, m/z 369 (M+1), 370 (M+2).

(Z)-2-[(E)-3-(4-methoxyphenyl)acrylamido]-3-phenyl-N-propylacrylamide(1912)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-benzylidene]-2-[(E)-4-methoxystyryl]oxazol-5(4H)-one (0.305 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 1912 was a whitesolid (303 mg, 83%), Mp 184-185° C. IR (KBr, ∇_(max) cm⁻¹) 3229, 2964,1650, 1625; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.62 (s, 1H), 8.12 (t,J=5.65 Hz, 1H), 7.51-7.62 (m, 4H), 7.45 (d, J=15.43 Hz, 1H), 7.38 (t,J=7.72 Hz, 2H), 7.31 (t, J=7.79 Hz, 1H), 7.02 (d, J=8.66 Hz, 2H), 6.97(s, 1H), 6.72 (d, J=15.81 Hz, 1H), 3.81 (s, 3H), 3.11 (q, J=6.40 Hz,2H), 1.48 (sxt, J=7.30 Hz, 2H), 0.87 (t, J=7.34 Hz, 3H); ¹³C NMR (214MHz, DMSO-d₆) δ_(C) 165.1, 164.9, 160.6, 139.7, 134.4, 130.7, 129.4,129.3, 128.6, 128.5, 127.3, 126.6, 119.0, 114.5, 55.4, 41.0, 39.8, 39.7,39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 11.5; LC/MS (ESI), RT=3.5 min, m/z365 (M+1).

(Z)-3-phenyl-N-propyl-2-[(E)-3-(p-tolyl)acrylamido]acrylamide (2012)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-benzylidene]-2-[(E)-4-methylstyryl]oxazol-5(4H)-one (0.289 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2012 was anoff-white solid (321 mg, 92%), Mp 175-176° C. IR (KBr, ν_(max) cm⁻¹)3296, 3172, 2967, 1646, 1610; ¹H NMR (850 MHz, DMSO-d₆) δ_(H) 9.65 (br.s., 1H), 8.11 (t, J=5.45 Hz, 1H), 7.48-7.57 (m, 4H), 7.46 (d, J=15.57Hz, 1H), 7.37-7.40 (m, 2H), 7.27-7.34 (m, 2H), 7.26 (d, J=7.78 Hz, 1H),6.98-7.01 (m, 1H), 6.82 (d, J=15.57 Hz, 1H), 3.09-3.13 (m, 2H), 1.48(td, J=7.20, 14.14 Hz, 2H), 0.87 (t, J=7.27 Hz, 3H); ¹³C NMR (214 MHz,DMSO-d₆) δ_(C) 165.2, 164.9, 140.1, 139.8, 134.4, 132.1, 130.6, 129.8,129.4, 128.7, 127.9, 127.5, 126.9, 120.6, 41.1, 39.8, 39.7, 39.6, 39.5,39.4, 39.3, 39.2, 22.5, 21.1, 11.6; LC/MS (ESI), RT=4.3 min, m/z 349(M+1).

(Z)-3-phenyl-N-propyl-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide (2112)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-benzylidene]-2-[(E)-2-(thien-3-yl)vinyl]oxazol-5(4H)-one (0.281g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2112 was anoff-white solid (263 mg, 77%), Mp 195-196° C. IR (KBr, ν_(max) cm⁻¹)3296, 3172, 2967, 1646, 1610; ¹H NMR (850 MHz, DMSO-d₆) δ_(H) 9.65 (s,1H), 8.10 (t, J=5.71 Hz, 1H), 7.61-7.68 (m, 2H), 7.53 (d, J=7.79 Hz,2H), 7.43 (d, J=3.11 Hz, 1H), 7.39 (t, J=7.79 Hz, 2H), 7.31 (t, J=7.79Hz, 1H), 7.14 (dd, J=3.37, 4.93 Hz, 1H), 6.99 (s, 1H), 6.62 (d, J=15.57Hz, 1H), 3.11 (q, J=6.75 Hz, 2H), 1.48 (sxt, J=7.27 Hz, 2H), 0.87 (t,J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 165.0, 164.4, 139.8,134.3, 133.0, 131.3, 130.5, 129.3, 128.6, 128.6, 128.5, 128.5, 126.9,120.3, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 11.5; LC/MS(ESI), RT=3.4 min, m/z 341 (M+1).

(Z)-2-cinnamamido-3-(4-fluorophenyl)-N-propylacrylamide (2212)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-fluorobenzylidene]-2-[(E)-styryl]oxazol-5(4H)— (0.293 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2212 was anoff-white solid (290 mg, 82%), Mp 192-193° C. IR (KBr, ν_(max) cm⁻¹)3360, 3026, 2950, 1650, 1610; ¹H NMR (DMSO-d₆) δ_(H) ppm 9.68 (s, 1H),8.12 (s, 1H), 7.56-7.67 (m, 4H), 7.50 (d, J=16.09 Hz, 1H), 7.44-7.47 (m,2H), 7.42 (d, J=7.78 Hz, 1H), 7.23 (t, J=8.82 Hz, 2H), 7.01 (s, 1H),6.86 (d, J=16.09 Hz, 1H), 3.12 (q, J=6.75 Hz, 2H), 1.45-1.52 (m, 2H),0.87 (t, J=7.53 Hz, 3H); LC/MS (ESI), RT=3.9 min, m/z 353 (M+1).

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-3-(4-fluorophenyl)-N-propylacrylamide(2312)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from2-[(E)-4-chlorostyryl]-4-[(Z)-4-fluorobenzylidene]oxazol-5(4H)-one(0.327 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2312was an off-white solid (341 mg, 88%), Mp 187-188° C. IR (KBr, ν_(max)cm⁻¹) 3360, 3002, 2949, 1650, 1610; ¹H NMR (850 MHz, DMSO-d₆) δ_(H) 9.69(s, 1H), 8.12 (t, J=5.71 Hz, 1H), 7.64 (d, J=8.30 Hz, 2H), 7.60 (dd,J=5.71, 8.82 Hz, 2H), 7.46-7.55 (m, 3H), 7.20-7.26 (m, 2H), 7.01 (s,1H), 6.85 (d, J=16.09 Hz, 1H), 3.11 (q, J=6.40 Hz, 2H), 1.48 (sxt,J=7.27 Hz, 2H), 0.87 (t, J=7.27 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆)δ_(C) 164.9, 164.4, 138.7, 134.3, 133.7, 131.5, 130.9, 130.1, 129.5,129.2, 125.9, 122.4, 115.6, 115.5, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4,39.3, 39.2, 22.4, 11.5; LC/MS (ESI), RT=5.1 min, m/z 387 (M+1), 388(M+2).

(Z)-3-(4-fluorophenyl)-2-[(E)-3-(4-methoxyphenyl)acrylamido]-N-propylacrylamide(2412)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-fluorobenzylidene)]-2-[(E)-4-methoxystyryl]oxazol-5(4H)-one(0.323 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2412was a white solid (355 mg, 93%), Mp 199-200° C. IR (KBr, ν_(max) cm⁻¹)3285, 2961, 1649, 1608; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.61 (s, 1H),8.12 (t, J=5.65 Hz, 1H), 7.56-7.61 (m, 4H), 7.45 (d, J=15.81 Hz, 1H),7.20-7.26 (m, 2H), 7.02 (d, J=8.66 Hz, 2H), 6.98 (s, 1H), 6.71 (d,J=15.81 Hz, 1H), 3.81 (s, 3H), 3.11 (q, J=6.65 Hz, 2H), 1.48 (sxt,J=7.30 Hz, 2H), 0.87 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆)δ_(C) 165.1, 164.9, 160.7, 139.9, 131.5, 131.0, 130.4, 129.5, 127.3,125.6, 119.0, 115.6, 115.5, 114.6, 55.4, 41.0, 39.8, 39.7, 39.6, 39.5,39.4, 39.3, 39.2, 22.4, 11.5; LC/MS (ESI), RT=3.8 min, m/z 383 (M+1).

(Z)-3-(4-fluorophenyl)-N-propyl-2-[(E)-3-(p-tolyl)acrylamido]acrylamide(2512)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-fluorobenzylidene]-2-[(E)-4-methylstyryl]oxazol-5(4H)-one(0.307 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2512was an off-white solid (289 mg, 79%), Mp 164-165° C. IR (KBr, ν_(max)cm⁻¹) 3302, 3208, 2970, 1649, 1640; ¹H NMR (850 MHz, DMSO-d₆) δ_(H) 9.64(s, 1H), 8.11 (t, J=5.71 Hz, 1H), 7.60 (dd, J=5.71, 8.82 Hz, 2H), 7.50(d, J=8.30 Hz, 2H), 7.46 (d, J=16.09 Hz, 1H), 7.26 d, J=8.30 Hz, 2H),7.20-7.25 (m, 2H), 7.00 (s, 1H), 6.81 (d, J=15.57 Hz, 1H), 3.12 (q,J=6.75 Hz, 2H), 2.35 (s, 3H), 1.48 (sxt, J=7.27 Hz, 2H), 0.87 (t, J=7.27Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 165.0, 164.8, 140.0, 139.7,132.0, 131.5, 131.0, 130.3, 129.7, 127.8, 125.7, 120.5, 115.6, 115.5,41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 21.1, 11.5; LC/MS(ESI), RT=3.7 min, m/z 367 (M+1).

(Z)-3-(4-fluorophenyl)-N-propyl-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide(2612)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-([(Z)-4-fluorobenzylidene]-2-[(E)-2-(thien-3-yl)vinyl]oxazol-5(4H)-one(0.299 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2612was a yellow solid (279 mg, 78%), Mp 142-143° C. IR (KBr, ν_(max) cm⁻¹)3302, 3143, 2961, 1650, 1610; ¹H NMR (850 MHz, DMSO-d₆) δ ppm 9.63 (s,1H) 8.11 (t, J=5.71 Hz, 1H) 7.61-7.68 (m, 2H) 7.58 (dd, J=8.04, 5.97 Hz,2H) 7.43 (d, J=3.11 Hz, 1H) 7.23 (t, J=8.56 Hz, 2H) 7.11-7.16 (m, 1H)7.00 (s, 1H) 6.60 (d, J=15.57 Hz, 1H) 3.11 (q, J=6.40 Hz, 2H) 1.48 (sxt,J=7.27 Hz, 2H) 0.87 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C)165.0, 164.4, 139.8, 133.1, 131.6, 131.5, 131.4, 130.9, 130.2, 128.6,128.5, 126.3, 125.9, 120.3, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3,39.2, 22.4, 11.5; LC/MS (ESI), RT=3.5 min, m/z 359 (M+1).

(Z)-2-cinnamamido-3-[4-(dimethylamino)phenyl]-N-propylacrylamide (2712)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-(dimethylamino)benzylidene]-2-[(E)-styryl]oxazol-5(4H)-one(0.318 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2712was an off-white solid (328 mg, 87%), Mp 207-208° C. IR (KBr, ν_(max)cm⁻¹) 3087, 2933, 1659; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.51 (s, 1H),7.91 (t, J=5.84 Hz, 1H), 7.63 (d, J=7.15 Hz, 2H), 7.45-7.52 (m, 3H),7.40-7.44 (m, 3H), 7.04 (s, 1H), 6.90 (d, J=15.81 Hz, 1H), 6.70 (d,J=9.03 Hz, 2H), 3.11 (q, J=6.75 Hz, 2H), 2.92 (s, 6H), 1.46 (sxt, J=7.23Hz, 2H), 0.86 (t, J=7.34 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C)169.6, 168.0, 142.5, 141.1, 135.8, 134.7, 131.2, 129.3, 129.0, 128.7,128.1, 127.5, 126.3, 111.0, 41.6, 40.7, 39.8, 39.7, 39.6, 39.5, 39.4,39.3, 39.2, 22.3, 11.4; LC/MS (ESI), RT=3.8 min, m/z 378 (M+1).

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-3-[4-(dimethylamino)phenyl]-N-propylacrylamide(2812)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from2-[(E)-4-chlorostyryl]-4-[(Z)-4-(dimethylamino)benzylidene]oxazol-5(4H)-one(0.353 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2812was a yellow solid (371 mg, 90%), Mp 243-244° C. IR (KBr, ν_(max) cm⁻¹)3067, 2912, 1648, 1609; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.53 (s, 1H),7.92 (t, J=6.02 Hz, 1H), 7.65 (d, J=8.66 Hz, 2H), 7.53 (d, J=8.28 Hz,2H), 7.50 (d, J=15.81 Hz, 1H), 7.42 (d, J=9.03 Hz, 2H), 7.04 (s, 1H),6.90 (d, J=15.81 Hz, 1H), 6.69 (d, J=9.03 Hz, 2H), 3.10 (q, J=6.40 Hz,2H), 2.93 (s, 6H), 1.46 (sxt, J=7.23 Hz, 2H), 0.86 (t, J=7.34 Hz, 3H);¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 166.5, 163.1, 140.1, 137.9, 134.6,133.4, 131.2, 129.4, 129.3, 128.8, 128.6, 128.2, 127.8, 127.5, 41.3,40.5, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 11.4; LC/MS (ESI),RT=5.9 min, m/z 412 (M+1), 413 (M+2).

(Z)-3-[4-(dimethylamino)phenyl]-2-[(E)-3-(4-methoxyphenyl)acrylamido]-N-propylacrylamide(2912)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-(dimethylamino)benzylidene]-2-[(E)-4-methoxystyryl]-oxazol-5(4H)-one(0.348 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 2912was a yellow solid (346 mg, 85%), Mp 236-237° C. IR (KBr, ν_(max) cm⁻¹)3070, 2952, 1647, 1603; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.41 (s, 1H),7.88 (t, J=5.83 Hz, 1H), 7.58 (d, J=9.04 Hz, 2H), 7.40-7.47 (m, 3H),7.01-7.04 (m, 3H), 6.75 (d, J=15.81 Hz, 1H), 6.69 (d, J=9.03 Hz, 2H),3.81 (s, 3H), 3.10 (q, J=6.40 Hz, 2H), 2.92 (s, 6H), 1.46 (sxt, J=7.30Hz, 2H), 0.86 (t, J=7.34 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C)164.3, 163.1, 158.6, 142.3, 134.5, 133.1, 131.9, 131.4, 129.4, 128.6,119.0, 114.6, 113.9, 113.2, 55.3, 41.5, 40.5, 39.8, 39.7, 39.6, 39.5,39.4, 39.3, 39.2, 22.1, 11.3; LC/MS (ESI), RT=3.7 min, m/z 408 (M+1).

(Z)-3-[4-(dimethylamino)phenyl]-N-propyl-2-[(E)-3-(p-tolyl)acrylamido]acrylamide(3012)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-(dimethylamino)benzylidene]-2-[(E)-4-methylstyryl]oxazol-5(4H)-one(0.332 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3012was a yellow solid (341 mg, 87%), Mp 218-219° C. IR (KBr, ν_(max) cm⁻¹)3268, 2952, 1647, 1601; ¹H NMR (850 MHz, DMSO-d₆) δ ppm 9.51 (br. s.,1H) 7.96 (t, J=5.97 Hz, 1H) 7.45-7.52 (m, 3H) 7.26 (m, J=7.78 Hz, 2H)7.21 (s, 1H) 7.04 (s, 1H) 6.99 (d, J=8.30 Hz, 1H) 6.84 (d, J=15.57 Hz,1H) 6.76 (d, J=8.30 Hz, 1H) 3.11 (q, J=6.57 Hz, 2H) 2.51 (br. s., 6H)2.34 (s, 3H) 1.47 (sxt, J=7.16 Hz, 2H) 0.85-0.87 (m, 3H); LC/MS (ESI),RT=5.0 min, m/z 392 (M+1).

(Z)-3-[4-(dimethylamino)phenyl]-N-propyl-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide(3112)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-(dimethylamino)benzylidene]-2-[(E)-2-(thien-3-yl)vinyl]-oxazol-5(4H)-one(0.324 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3112was an off-white solid (352 mg, 92%), Mp 208-209° C. IR (KBr, ν_(max)cm⁻¹) 3296, 2948, 1643, 1605; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.47 (s,1H), 7.90 (t, J=5.83 Hz, 1H), 7.61-7.68 (m, 2H), 7.44 (d, J=3.39 Hz,1H), 7.40 (d, J=9.04 Hz, 2H), 7.15 (dd, J=3.58, 5.08 Hz, 1H), 7.03 (s,1H), 6.70 (d, J=9.03 Hz, 2H), 6.64 (d, J=15.81 Hz, 1H), 3.09 (q, J=6.40Hz, 2H), 2.93 (s, 6H), 1.46 (sxt, J=7.30 Hz, 2H), 0.85 (t, J=7.34 Hz,3H); LC/MS (ESI), RT=3.5 min, m/z 384 (M+1).

(Z)-2-cinnamamido-3-(4-hydroxy-3-methoxyphenyl)-N-propylacrylamide(3212)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-hydroxy-3-methoxybenzylidene]-2-[(E)-styryl]oxazol-5(4H)-one(0.321 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3212was an off-white solid (323 mg, 85%), Mp 199-200° C. IR (KBr, ν_(max)cm⁻¹) 3292, 2971, 1650, 1610; ¹H-NMR (600 MHz, DMSO-d₆) δ_(H) 8.02 (br.s., 1H), 7.61 (d, J=7.15 Hz, 2H), 7.39-7.55 (m, 4H), 7.21 (s, 1H), 7.04(s, 1H), 6.91 (d, J=16.19 Hz, 1H), 6.77 (d, J=8.28 Hz, 1H), 3.69 (s,3H), 3.11 (q, J=6.65 Hz, 2H), 1.47 (sxt, J=7.15 Hz, 2H), 0.86 (t, J=7.34Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 165.2, 164.8, 147.5, 147.3,139.8, 134.9, 129.9, 129.2, 128.6, 127.7, 127.2, 125.5, 123.8, 121.8,115.5, 112.9, 55.4, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2,22.5, 11.5; LC/MS (ESI), RT=2.9 min, m/z 381 (M+1).

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-3-(4-hydroxy-3-methoxyphenyl)-N-propylacrylamide(3312)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from2-[(E)-4-chlorostyryl]-4-[(Z)-4-hydroxy-3-methoxybenzylidene]-oxazol-5(4H)-one(0.355 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3312was a yellow solid (356 mg, 86%), Mp 136-137° C. IR (KBr, ν_(max) cm⁻¹)3566, 2988, 2902, 1650; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.61 (br. s.,1H), 8.01 (t, J=5.83 Hz, 1H), 7.64 (d, J=8.28 Hz, 2H), 7.48-7.56 (m,3H), 7.20 (d, J=2.26 Hz, 1H), 7.04 (s, 1H), 6.99 (dd, J=1.88, 8.28 Hz,1H), 6.90 (d, J=16.19 Hz, 1H), 6.76 (d, J=8.28 Hz, 1H), 3.68 (s, 3H),3.11 (q, J=6.65 Hz, 2H), 1.47 (sxt, J=7.23 Hz, 2H), 0.86 (t, J=7.34 Hz,3H); LC/MS (ESI), RT=3.4 min, m/z 415 (M+1), 416 (M+2).

(Z)-3-(4-hydroxy-3-methoxyphenyl)-2-[(E)-3-(4-methoxyphenyl)acrylamido]-N-propylacrylamide(3412)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-hydroxy-3-methoxybenzylidene]-2-[(E)-4-methoxystyryl]-oxazol-5(4H)-one(0.351 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3412was an orange solid (332 mg, 81%), Mp 180-181° C. IR (KBr, ν_(max) cm⁻¹)3567, 2989, 2955, 1655; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.49 (br. s.,1H), 7.97 (t, J=6.02 Hz, 1H), 7.56 (d, J=8.28 Hz, 2H), 7.46 (d, J=15.81Hz, 1H), 7.21 (d, J=1.88 Hz, 1H), 7.00-7.03 (m, 3H), 6.99 (dd, J=1.88,8.28 Hz, 1H), 6.77 (d, J=6.78 Hz, 1H), 6.75 (s, 1H), 3.81 (s, 3H), 3.68(s, 3H), 3.11 (q, J=6.65 Hz, 2H), 1.47 (sxt, J=7.23 Hz, 2H), 0.86 (t,J=7.34 Hz, 3H); LC/MS (ESI), RT=2.7 min, m/z 411 (M+1).

(Z)-3-(4-hydroxy-3-methoxyphenyl)-N-propyl-2-[(E)-3-(p-tolyl)acrylamido]acrylamide(3512)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-hydroxy-3-methoxybenzylidene]-2-[(E)-4-methylstyryl]-oxazol-5(4H)-one(0.335 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3512was a yellow solid (296 mg, 75%), Mp 183-184° C. IR (KBr, ν_(max) cm⁻¹)3566, 3213, 2947, 1663; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.54 (s, 1H),9.39 (br. s., 1H), 8.00 (t, J=5.65 Hz, 1H), 7.44-7.53 (m, 3H), 7.27 (d,J=7.91 Hz, 2H), 7.21 (s, 1H), 7.03 (s, 1H), 6.99 (dd, J=1.88, 8.28 Hz,1H), 6.85 (d, J=15.81 Hz, 1H), 6.76 (d, J=8.28 Hz, 1H), 3.68 (s, 3H),3.10 (q, J=6.65 Hz, 2H), 2.35 (s, 3H), 1.47 (sxt, J=7.23 Hz, 2H), 0.86(t, J=7.34 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 165.1, 164.9,147.5, 147.3, 139.7, 139.6, 132.1, 129.7, 128.5, 127.7, 127.3, 125.5,123.7, 120.8, 115.4, 112.9, 55.3, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4,39.3, 39.2, 22.5, 11.5; LC/MS (ESI), RT=3.3 min, m/z 395 (M+1).

(Z)-3-(4-hydroxy-3-methoxyphenyl)-N-propyl-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide(3612)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-hydroxy-3-methoxybenzylidene]-2-[(E)-2-(thien-3-yl)vinyl]-oxazol-5(4H)-one(0.327 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3612was a yellow solid (340 mg, 88%), Mp 192-93° C. IR (KBr, ν_(max) cm⁻¹)3560, 3105, 2954, 1648, 1603; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.54 (br.s., 1H), 7.99 (t, J=5.83 Hz, 1H), 7.63-7.68 (m, 2H), 7.43 (d, J=3.39 Hz,1H), 7.19 (d, J=1.88 Hz, 1H), 7.13-7.16 (m, 1H), 7.04 (s, 1H), 6.98 (dd,J=1.88, 8.28 Hz, 1H), 6.76 (d, J=7.91 Hz, 1H), 6.66 (d, J=15.81 Hz, 1H),3.69 (s, 3H), 3.11 (q, J=6.65 Hz, 2H), 1.46 (sxt, J=7.23 Hz, 2H), 0.86(t, J=7.34 Hz, 3H); LC/MS (ESI), RT=2.7 min, m/z 387 (M+1).

(Z)-2-cinnamamido-3-(1H-indol-3-yl)-N-propylacrylamide (3712)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-[(1H-indol-3-yl)methylene]-2-[(E)-styryl]oxazol-5(4H)-one (0.314g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3712 was awhite solid (287 mg, 77%), Mp 249-250° C. IR (KBr, ν_(max) cm⁻¹) 3401,3057, 2956, 1650, 1608; NMR (600 MHz, DMSO-d₆) δ_(H) 11.56 (br. s., 1H),9.46 (s, 1H), 7.98 (t, J=5.83 Hz, 1H), 7.74 (d, J=7.91 Hz, 1H),7.63-7.67 (m, 3H), 7.42-7.55 (m, 6H), 7.17 (t, J=7.53 Hz, 1H), 7.12 (t,J=7.53 Hz, 1H), 6.98 (d, J=15.81 Hz, 1H), 3.14 (q, J=6.53 Hz, 2H), 1.50(sxt, J=7.23 Hz, 2H), 0.88 (t, J=7.34 Hz, 3H); ¹³C NMR (214 MHz,DMSO-d₆) δ_(C) 164.5, 164.2, 141.5, 135.5, 134.8, 129.6, 129.3, 129.1,129.0, 128.7, 127.9, 124.9, 124.6, 122.1, 120.0, 118.2, 111.9, 110.0,41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.3, 11.5; LC/MS (ESI),RT=3.3 min, m/z 374 (M+1).

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-3-(1H-indol-3-yl)-N-propylacrylamide(3812)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-[(1H-indol-3-yl)methylene]-2-[(E)-4-chlorostyryl]oxazol-5(4H)-one(0.348 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3812was an orange solid (330 mg, 81%), Mp 210-211° C. IR (KBr, ν_(max) cm⁻¹)3208, 1650, 1615; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 11.56 (br. s., 1H),9.47 (s, 1H), 7.98 (t, J=5.83 Hz, 1H), 7.73 (d, J=7.91 Hz, 1H), 7.68 (d,J=8.66 Hz, 2H), 7.65 (d, J=2.63 Hz, 1H), 7.50-7.56 (m, 4H), 7.43 (d,J=7.91 Hz, 1H), 7.17 (t, J=7.15 Hz, 1H), 7.12 (t, J=7.34 Hz, 1H), 6.98(d, J=15.81 Hz, 1H), 3.14 (q, J=6.53 Hz, 2H), 1.50 (sxt, J=7.30 Hz, 2H),0.88 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 165.7, 165.3,140.5, 138.1, 135.7, 134.2, 132.3, 132.2, 130.7, 130.6, 130.5, 130.1,129.8, 125.1, 123.9, 122.7, 113.7, 111.5, 110.1, 41.6, 39.8, 39.7, 39.6,39.5, 39.4, 39.3, 39.2, 23.0, 11.6; LC/MS (ESI), RT=4.2 min, m/z 408(M+1), 409 (M+2).

(Z)-3-(1H-indol-3-yl)-2-[(E)-3-(4-methoxyphenyl)acrylamido]-N-propylacrylamide(3912)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-[(1H-indol-3-yl)methylene]-2-[(E)-4-methoxystyryl]oxazol-5(4H)-one(0.344 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 3912was an orange solid (335 mg, 83%), Mp 225-226° C. IR (KBr, ν_(max) cm⁻¹)3181, 1650, 1615; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 11.55 (br. s., 1H),9.36 (s, 1H), 7.95 (t, J=5.83 Hz, 1H), 7.73 (d, J=7.91 Hz, 1H), 7.64 (d,J=2.26 Hz, 1H), 7.60 (d, J=8.66 Hz, 2H), 7.46-7.50 (m, 2H), 7.43 (d,J=7.91 Hz, 1H), 7.17 (t, J=7.53 Hz, 1H), 7.12 (t, J=7.53 Hz, 1H), 7.03(d, J=8.66 Hz, 2H), 6.84 (d, J=15.81 Hz, 1H), 3.82 (s, 3H), 3.14 (q,J=6.27 Hz, 2H), 1.49 (sxt, J=7.23 Hz, 2H), 0.88 (t, J=7.34 Hz, 3H); ¹³CNMR (214 MHz, DMSO-d₆) δ_(C) 166.0, 164.3, 160.6, 141.5, 138.5, 137.1,132.3, 131.9, 131.4, 129.2, 123.5, 122.2, 121.0, 120.6, 114.6, 113.9,112.5, 112.3, 55.8, 41.5, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2,22.6, 11.5; LC/MS (ESI), RT=3.2 min, m/z 404 (M+1).

(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(p-tolyl)acrylamido]acrylamide(4012)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-[(1H-indol-3-yl)methylene]-2-[(E)-4-methylstyryl]oxazol-5(4H)-one(0.328 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4012was a yellow solid (345 mg, 89%), Mp 192-193° C. IR (KBr, ν_(max) cm⁻¹)3220, 2950, 1651, 1595; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 11.56 (br. s.,1H), 9.42 (s, 1H), 7.97 (t, J=5.83 Hz, 1H), 7.73 (d, J=7.91 Hz, 1H),7.65 (s, 1H), 7.54 (d, J=7.91 Hz, 2H), 7.47-7.52 (m, 2H), 7.43 (d,J=7.91 Hz, 1H), 7.28 (d, J=7.91 Hz, 2H), 7.17 (t, J=7.53 Hz, 1H), 7.12(t, J=7.53 Hz, 1H), 6.93 (d, J=15.81 Hz, 1H), 3.14 (q, J=6.40 Hz, 2H),2.36 (s, 3H), 1.50 (sxt, J=7.30 Hz, 2H), 0.88 (t, J=7.53 Hz, 3H); ¹³CNMR (214 MHz, DMSO-d₆) δ_(C) 165.1, 164.6, 141.5, 139.6, 135.5, 129.8,129.6, 129.2, 127.9, 127.7, 127.6, 122.2, 121.3, 121.1, 118.2, 112.5,111.9, 110.0, 40.9, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.6,21.0, 11.5; LC/MS (ESI), RT=3.7 min, m/z 388 (M+1).

(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide(4112)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-[(1H-indol-3-yl)methylene]-2-[(E)-2-(thien-3-yl)vinyl]oxazol-5(4H)-one(0.320 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4112was a yellow solid (326 mg, 86%), Mp 244-245° C. IR (KBr, ν_(max) cm⁻¹)3567, 3997, 2950, 1650; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 11.56 (br. s.,1H), 9.41 (s, 1H), 7.96 (t, J=5.83 Hz, 1H), 7.72 (d, J=7.91 Hz, 1H),7.65-7.70 (m, 2H), 7.64 (d, J=1.88 Hz, 1H), 7.50 (s, 1H), 7.42-7.46 (m,2H), 7.14-7.19 (m, 2H), 7.12 (t, J=7.53 Hz, 1H), 6.73 (d, J=15.43 Hz,1H), 3.13 (q, J=6.40 Hz, 2H), 1.49 (sxt, J=7.23 Hz, 2H), 0.88 (t, J=7.53Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 163.8, 163.2, 139.7, 138.1,136.5, 136.2, 133.4, 133.3, 132.4, 129.1, 128.3, 123.6, 122.9, 122.2,121.1, 120.6, 112.3, 107.4, 41.4, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3,39.2, 22.3, 11.3; LC/MS (ESI), RT=3.1 min, m/z 380 (M+1).

(Z)-2-cinnamamido-N-propyl-3-(pyridin-3-yl)acrylamide (4212)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-(pyridin-3-ylmethylene)-2-[(E)-styryl]oxazol-5(4H)-one (0.276 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4212 was a whitesolid (248 mg, 74%), Mp 189-190° C. IR (KBr, ν_(max) cm⁻¹) 3258, 2964,1649, 1610; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.82 (s, 1H), 8.70 (d,J=2.26 Hz, 1H), 8.47 (dd, J=1.69, 4.71 Hz, 1H), 8.25 (t, J=5.65 Hz, 1H),7.92 (td, J=1.74, 8.19 Hz, 1H), 7.60-7.66 (m, 2H), 7.51 (d, J=15.81 Hz,1H), 7.39-7.49 (m, 4H), 7.00 (s, 1H), 6.86 (d, J=16.19 Hz, 1H), 3.13 (q,J=6.40 Hz, 2H), 1.49 (sxt, J=7.30 Hz, 2H), 0.88 (t, J=7.34 Hz, 3H); ¹³CNMR (214 MHz, DMSO-d₆) δ_(C) 164.7, 164.5, 150.1, 148.9, 140.3, 135.9,134.7, 132.2, 130.5, 129.9, 129.1, 127.8, 123.7, 123.2, 121.4, 41.0,39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 11.5; LC/MS (ESI),RT=2.7 min, m/z 336 (M+1).

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-N-propyl-3-(pyridin-3-yl)acrylamide(4312)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-2-[(E)-4-chlorostyryl]-4-(pyridin-3-ylmethylene)oxazol-5(4H)-one(0.310 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4312was a white solid (288 mg, 78%), Mp 191-192° C. IR (KBr, ν_(max) cm⁻¹)3261, 2964, 1649, 1609; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.83 (br. s.,1H), 8.70 (d, J=2.26 Hz, 1H), 8.47 (dd, J=1.69, 4.71 Hz, 1H), 8.25 (t,J=5.46 Hz, 1H), 7.92 (d, J=7.91 Hz, 1H), 7.63 (d, J=7.15 Hz, 2H), 7.51(d, J=15.81 Hz, 1H), 7.44-7.48 (m, 2H), 7.40-7.44 (m, 2H), 7.00 (s, 1H),6.86 (d, J=15.81 Hz, 1H), 3.13 (q, J=6.53 Hz, 2H), 1.49 (sxt, J=7.30 Hz,2H), 0.88 (t, J=7.34 Hz, 3H).

(Z)-2-[(E)-3-(4-methoxyphenyl)acrylamido]-N-propyl-3-(pyridin-3-yl)acrylamide(4412)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-2-[(E)-4-methoxystyryl]-4-(pyridin-3-ylmethylene)oxazol-5(4H)-one(0.306 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4412was a white solid (300 mg, 82%), Mp 185-186° C. IR (KBr, ν_(max) cm⁻¹)3273, 3220, 2964, 1648, 1616; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.72 (s,1H), 8.69 (s, 1H), 8.47 (dd, J=1.69, 4.71 Hz, 1H), 8.22 (t, J=5.27 Hz,1H), 7.91 (dd, J=1.32, 8.09 Hz, 1H), 7.57 (d, J=8.66 Hz, 2H), 7.46 (d,J=15.43 Hz, 1H), 7.41 (dd, J=4.71, 8.09 Hz, 1H), 7.02 (d, J=8.66 Hz,2H), 6.97 (s, 1H), 6.71 (d, J=15.81 Hz, 1H), 3.81 (s, 3H), 3.13 (q,J=6.53 Hz, 2H), 1.49 (sxt, J=7.23 Hz, 2H), 0.88 (t, J=7.34 Hz, 3H), ¹³CNMR (214 MHz, DMSO-d₆) δ_(C) 164.8, 164.4, 160.7, 150.1, 148.9, 140.1,135.9, 132.3, 130.6, 129.5, 127.3, 123.7, 123.0, 118.8, 114.6, 55.4,41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 11.5; LC/MS (ESI),RT=2.7 min, m/z 366 (M+1).

(Z)—N-propyl-3-(pyridin-3-yl)-2-[(E)-3-(p-tolyl)acrylamido]acrylamide(4512)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-2-[(E)-4-methylstyryl]-4-(pyridin-3-ylmethylene)oxazol-5(4H)-one(0.290 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4512was a white solid (272 mg, 78%), Mp 190-191° C. IR (KBr, ν_(max) cm⁻¹)3270, 2967, 1648, 1608; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.77 (s, 1H),8.69 (d, J=1.88 Hz, 1H), 8.47 (dd, J=1.69, 4.71 Hz, 1H), 8.23 (t, J=5.83Hz, 1H), 7.92 (td, J=1.60, 8.09 Hz, 1H), 7.52 (d, J=7.91 Hz, 2H), 7.47(d, J=15.81 Hz, 1H), 7.41 (dd, J=4.33, 8.09 Hz, 1H), 7.27 (d, J=7.91 Hz,2H), 6.98 (s, 1H), 6.80 (d, J=15.81 Hz, 1H), 3.12 (q, J=6.65 Hz, 2H),2.35 (s, 3H), 1.49 (sxt, J=7.30 Hz, 2H), 0.88 (t, J=7.34 Hz, 3H); ¹³CNMR (214 MHz, DMSO-d₆) δ_(C) 164.7, 164.6, 150.1, 148.9, 140.3, 139.8,135.9, 132.3, 132.0, 130.6, 129.7, 127.8, 123.7, 123.1, 120.4, 41.1,39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.4, 21.1, 11.5; LC/MS (ESI),RT=3.1 min, m/z 350 (M+1).

(Z)—N-propyl-3-(pyridin-3-yl)-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide(4612)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from(Z)-4-(pyridin-3-ylmethylene)-2-[(E)-2-(thien-3-yl)vinyl]oxazol-5(4H)-one(0.282 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4612was an off-white solid (300 mg, 88%), Mp 182-183° C. IR (KBr, ν_(max)cm⁻¹) 3270, 2967, 1647, 1605; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.78 (br.s., 1H), 8.68 (d, J=1.88 Hz, 1H), 8.47 (dd, J=1.51, 4.89 Hz, 1H), 8.23(t, J=5.84 Hz, 1H), 7.91 (td, J=1.79, 8.09 Hz, 1H), 7.62-7.70 (m, 2H),7.39-7.47 (m, 2H), 7.15 (dd, J=3.58, 5.08 Hz, 1H), 6.99 (s, 1H), 6.60(d, J=15.81 Hz, 1H), 3.12 (q, J=6.65 Hz, 2H), 1.49 (sxt, J=7.23 Hz, 2H),0.88 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 164.6, 164.2,150.1, 148.9, 139.7, 135.9, 133.3, 132.1, 131.5, 131.5, 130.5, 128.6,123.7, 123.2, 120.1, 41.0, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2,22.3, 11.5; LC/MS (ESI), RT=2.8 min, m/z 342 (M+1).

(Z)-2-cinnamamido-3-(4-nitrophenyl)-N-propylacrylamide (4712)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-nitrobenzylidene]-2-[(E)-styryl]oxazol-5(4H)-one (0.320 g, 1mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4712 was a whitesolid (341 mg, 90%), Mp 211-212° C. IR (KBr, ν_(max) cm⁻¹) 3294, 2958,1647, 1619; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.94 (br. s., 1H), 8.34 (t,J=5.84 Hz, 1H), 8.23 (d, J=8.66 Hz, 2H), 7.77 (d, J=9.03 Hz, 2H), 7.62(d, J=7.15 Hz, 2H), 7.51 (d, J=15.81 Hz, 1H), 7.43-7.48 (m, 3H), 6.96(s, 1H), 6.86 (d, J=16.19 Hz, 1H), 3.13 (q, J=6.65 Hz, 2H), 1.50 (sxt,J=7.30 Hz, 2H), 0.89 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆)δ_(C) 164.7, 164.3, 146.4, 141.7, 140.5, 134.7, 133.8, 130.2, 130.0,129.1, 127.8, 123.7, 123.0, 121.3, 41.1, 39.8, 39.7, 39.6, 39.5, 39.4,39.3, 39.2, 22.3, 11.5; LC/MS (ESI), RT=3.7 min, m/z 380 (M+1).

(Z)-2-[(E)-3-(4-chlorophenyl)acrylamido]-3-(4-nitrophenyl)-N-propylacrylamide(4812)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from2-[(E)-4-chlorostyryl]-4-[(Z)-4-nitrobenzylidene]oxazol-5(4H)-one (0.355g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4812 was ayellow solid (363 mg, 88%), Mp 199-200° C. IR (KBr, ν_(max) cm⁻¹) 3070,2967, 1648, 1622, 1530, 1350; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.95 (s,1H), 8.34 (t, J=5.46 Hz, 1H), 8.23 (d, J=9.04 Hz, 2H), 7.77 (d, J=9.04Hz, 2H), 7.65 (d, J=8.66 Hz, 2H), 7.50-7.55 (m, 3H), 6.97 (s, 1H), 6.86(d, J=15.81 Hz, 1H), 3.13 (q, J=6.53 Hz, 2H), 1.50 (sxt, J=7.23 Hz, 2H),0.89 (t, J=7.53 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 164.7, 164.1,146.4, 141.7, 139.1, 134.4, 133.7, 133.7, 130.2, 129.5, 129.2, 123.7,123.1, 122.1, 41.1, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.3,11.5; LC/MS (ESI), RT=4.9 min, m/z 414 (M+1), 415 (M+2).

(Z)-2-[(E)-3-(4-methoxyphenyl)acrylamido]-3-(4-nitrophenyl)-N-propylacrylamide(4912)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from2-[(E)-4-methoxystyryl]-4-[(Z)-4-nitrobenzylidene]oxazol-5(4H)-one(0.350 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 4912was a yellow solid (348 mg, 85%), Mp 235-236° C. IR (KBr, ν_(max) cm⁻¹)3264, 2967, 1644, 1608, 1520, 1335; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.84(br. s., 1H), 8.31 (t, J=5.65 Hz, 1H), 8.23 (d, J=9.04 Hz, 2H), 7.77 (d,J=8.66 Hz, 2H), 7.57 (d, J=8.66 Hz, 2H), 7.46 (d, J=15.81 Hz, 1H), 7.02(d, J=8.66 Hz, 2H), 6.93 (s, 1H), 6.71 (d, J=15.81 Hz, 1H), 3.81 (s,3H), 3.13 (q, J=6.40 Hz, 2H), 1.50 (sxt, J=7.23 Hz, 2H), 0.89 (t, J=7.53Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ_(C) 164.8, 164.6, 160.7, 146.3,141.8, 140.3, 134.0, 130.1, 129.5, 127.2, 123.7, 122.6, 118.7, 114.5,55.4, 41.1, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.3, 11.5.

(Z)-3-(4-nitrophenyl)-N-propyl-2-[(E)-3-(p-tolyl)acrylamido]acrylamide(5012)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from2-[(E)-4-methylstyryl]-4-[(Z)-4-nitrobenzylidene]oxazol-5(4H)-one (0.334g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 5012 was ayellow solid (350 mg, 89%), Mp 206-207° C. IR (KBr, ν_(max) cm⁻¹) 3257,2964, 1647, 1621, 1515, 1340; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.89 (s,1H), 8.33 (t, J=5.65 Hz, 1H), 8.23 (d, J=9.04 Hz, 2H), 7.77 (d, J=9.04Hz, 2H), 7.45-7.52 (m, 3H), 7.27 (d, J=7.91 Hz, 2H), 6.95 (s, 1H), 6.80(d, J=15.81 Hz, 1H), 3.13 (q, J=6.53 Hz, 2H), 2.35 (s, 3H), 1.50 (sxt,J=7.30 Hz, 2H), 0.89 (t, J=7.34 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆)δ_(C) 164.8, 164.5, 146.4, 141.8, 140.5, 139.9, 133.9, 131.9, 130.1,129.7, 127.9, 123.7, 122.8, 120.3, 41.1, 39.8, 39.7, 39.6, 39.5, 39.4,39.3, 39.2, 22.3, 21.1, 11.5; LC/MS (ESI), RT=5.8 min, m/z 394 (M+1).

(Z)-3-(4-nitrophenyl)-N-propyl-2-[(E)-3-(thien-2-yl)acrylamido]acrylamide(5112)

This compound was prepared according to the procedure described for thesynthesis of 1503 starting from4-[(Z)-4-nitrobenzylidene]-2-[(E)-2-(thien-3-yl)vinyl]oxazol-5(4H)-one(0.326 g, 1 mmol) and n-propylamine (0.16 mL, 2 mmol). The product 5112was a yellow solid (320 mg, 83%), Mp 187-188° C. IR (KBr, ν_(max) cm⁻¹)3272, 2964, 1650, 1609, 1510, 1335; ¹H NMR (600 MHz, DMSO-d₆) δ_(H) 9.89(s, 1H), 8.32 (t, J=5.65 Hz, 1H), 8.24 (d, J=9.04 Hz, 2H), 7.76 (d,J=9.03 Hz, 2H), 7.64-7.69 (m, 2H), 7.45 (d, J=3.39 Hz, 1H), 7.15 (dd,J=3.58, 5.08 Hz, 1H), 6.95 (s, 1H), 6.60 (d, J=15.43 Hz, 1H), 3.12 (q,J=6.40 Hz, 2H), 1.50 (sxt, J=7.30 Hz, 2H), 0.89 (t, J=7.53 Hz, 3H); ¹³CNMR (214 MHz, DMSO-d₆) δ_(C) 164.7, 164.1, 146.4, 141.7, 139.7, 133.7,133.6, 131.6, 130.1, 128.7, 128.6, 123.7, 123.0, 120.0, 41.1, 39.8,39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 22.3, 11.5; LC/MS (ESI), RT=4.9 min,m/z 386 (M+1).

Biological Screening Methods

Materials for Cell Culture

Different cell lines were originally purchased from American typeculture collection (ATCC, Wesel, Germany) and grown in the tissueculture lab of the Egyptian company for production of vaccines, sera anddrugs (Vacsera, Giza, Egypt). The cells were transferred to ourlaboratory and maintained in the appropriate media as following.HCT-116, CACO-2, and HT-29 (human colon cancer cell lines) weremaintained in Roswell Park Memorial Institute medium (RPMI1640)(Invitrogen, Carlsbad, Calif.). The mouse skin fibroblasts (C-166) andBaby Hamster Kidney fibroblasts (BHK) were grown in Dulbecco ModifiedEagle's medium (DMEM). Both media were supplemented with 1% of 100 mg/mLof streptomycin, 100 units/mL of penicillin and 10% of heat-inactivatedfetal bovine serum (Invitrogen, Carlsbad, Calif.) in a humidified, 5%(v/v) CO2 atmosphere at 37° C.

Cytotoxicity Assay

The sulforhodamine B (SRB) assays were performed according to Skehan,P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R., New colorimetriccytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst1990, 82 (13), 1107-12, incorporated herein by reference in itsentirety. Briefly, exponentially growing cells were trypsinized, countedand seeded at the appropriate densities (5000 cells/100 μL/well) into96-well microtiter plates. Cells were incubated in a humidifiedatmosphere at 37° C. for 24 h. Then, the cells were exposed to differentcompounds at the desired concentrations, (0.01, 0.1, 1, 10, and 100 μM)or to 1% dimethyl sulfoxide (DMSO) for 72 h. At the end of the treatmentperiod, the media were removed, and the cells were fixed with 10%trichloroacetic acid at 4° C. for 1 hr. Following, the cells were washedwith tap water four times and incubated with SRB 0.4% for 30 min. Excessdye was removed by washing repeatedly with 1% (vol/vol) acetic acid. Theprotein-bound dye was dissolved in 10 mM Tris base solution for (opticaldensity) OD determination at 510 nm using a SpectraMax plus MicroplateReader (Molecular Devices, Calif.). Cell viability was expressedrelative to the untreated control cells.

Nuclear Fragmentation by DAPI Staining

Cells were cultured on sterile 22 mm² cover slips (Harvard Apparatus,Mass., USA) in sterile six well plates at a density of 2×10⁵ cells/well.24 h after seeding, cells were exposed to IC₅₀ of the tested compound infresh medium for 24 h. At the end of the exposure, cells attached tocover slips were washed with phosphate-buffered saline (PBS) and fixedwith 3.7% paraformaldhyde for 10 min, permeabilized with 0.25% TritonX-100 in tris-buffered saline-Tween 20 (TBST) containing 0.01% Tween 20for 10 min, and blocked for 1 hr with 5% goat serum in TBST. The fixedand permeabilized cells nuclei were denatured with 2 N HCl (300 μl) for10 min, washed three times more, and treated with 0.1 μg/ml4′,6′-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma-Aldrich,St. Louis, Mo., USA) (1:1000) in (phosphate-buffered saline-Tween 20(PBST) for 1 hr. After staining, the cells were washed twice with PBS.The cover slips were then mounted on a glass slide with anti-fademounting medium and viewed with an epifluorescence microscope, Leica, DM5500 B (Leica, Buffalo Grove, Ill., USA) at a magnification of 60×, anddata were captured digitally and quantified using the microscopeprovided software.

Cell Morphology

Cells were cultured on sterile 22 mm² cover slips (Harvard Apparatus,Mass., USA) in sterile six well plates at a density of 2×10⁵ cells/well.24 h after seeding, cells were exposed to IC₅₀ of the tested compoundsin fresh medium for 24 h. At the end of the exposure, cells attached tocover slips were washed with PBS and visualized under leica lightmicroscope (Leica, Buffalo Grove, Ill., USA).

Cell Cycle Analysis

To analyze the DNA content by flow cytometry, HCT-116 cells were seededat a density of 3×10⁶ cell/T 75 flask for 24 h and then exposed todifferent compounds at their IC₅₀ values for 24 h. The cells werecollected by trypsinization, washed with phosphate buffered saline (PBS)and fixed in ice-cold absolute alcohol. Thereafter, cells were stainedusing Cycletest™ Plus DNA Reagent Kit (BD Biosciences, San Jose, Calif.)according to the manufacturer's instructions. Cell cycle distributionwas determined using a FACS Calibur flow cytometer (BD Biosciences, SanJose, Calif.).

Primary (1°) Colonosphere Formation Assay

For primary sphere formation, cells were plated in nontreated, lowadhesion, 96 wells plate at the concentration of 100 cells/100 μL/wellin stem cell media (SCM) that consisted of DMEM:F12:AA (Gibco),supplemented with 1×B27 (Gibco), 20 ng/mL epidermal growth factor, and10 ng/mL fibroblast growth factor (Sigma). After 4 h of incubation,vehicle (control) or 3712 and 4112 at the desired concentrations wereadded to each well (at least in triplicates for each sample). On dayfive, numbers of spheres ranging from 50 to 150 mm in diameter werecounted using phase contrast microscope and percent inhibition wascalculated compared to control.

Determination of ROS Accumulation

To determine the effect of the newly synthesized compounds on thecellular redox status, two different free radical sensitive probedichlorofluorescin diacetate (DCFDA) was used. Cells were cultured onsterile 22 mm² cover slips (Harvard Apparatus, Mass., USA) in sterilesix well plates at a density of 2×105 cells/well. 24 h after seeding,cells were exposed to IC₅₀ of the tested compounds in fresh medium for24 h. At the end of the exposure, cells attached to cover slips werewashed thrice with PBS and incubated with DCFDA 10 μM for 30 min at 37°C. in the dark. Thereafter, cells were washed thrice with PBS and thecover slips were then mounted on a glass slide with anti-fade mountingmedium containing 4′,6′-diamidino-2-phenylindole, dihydrochloride (DAPI)(Sigma-Aldrich, St. Louis, Mo., USA), which was used as counter stainand viewed with an epifluorescence microscope, Leica, DM 5500 B (Leica,Buffalo Grove, Ill., USA) at a magnification of 60×. Data were captureddigitally and quantified using the microscope provided software.

Apoptotic Cell Determination

Apoptosis was determined by staining cells with Annexin V-fluoresceinisothiocyanate (FITC) and counterstaining with propidium iodide (PI)using the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, SanDiego, Calif., USA) according to the manufacturer's instructions.Briefly, 4×10⁶ cell/T 75 flask were exposed to the IC₅₀ of testedcompound for 24 and 48 h. The cells were collected by trypsinization and0.5×10⁶ cells were washed twice with phosphate-buffered saline (PBS) andstained with 5 μl Annexin V-FITC and 5 μl PI in 1× binding buffer (BDBiosciences, San Jose, Calif., USA) for 15 min at room temperature inthe dark. Analyses were performed using FACS Calibur flow cytometer (BDBiosciences, San Jose, Calif., USA).

Determination of Acute Oral Toxicity (LD₅₀)

A. Animals and Compounds

The experiment was conducted on 12 healthy Swiss albino mice (males andfemales) weighing 22-27 g and aged 8 to 10 weeks obtained from theAnimal Station, Pharmacology Dept, Faculty of Pharmacy, King AbdulazizUniversity, Jeddah. See Shoieb, S. M.; Esmat, A.; Khalifa, A. E.;Abdel-Naim, A. B., Chrysin attenuates testosterone-induced benignprostate hyperplasia in rats. Food and Chemical Toxicology 2018, 111,650-659, incorporated herein by reference in its entirety. All animalswere kept at the regulated temperature (average 23° C.), air quality(Central air conditioning) and light (12-h light/dark cycles). Animalswere provided free access to food pellets ad libitum and water. Theexperimental procedure was approved by the Research Ethics Committee,Faculty of Pharmacy, King Abdulaziz University prior to starting thelaboratory work. Animals were humanely treated according tointernational and scientific principles. Changes other than vitality ofthe animals such as behavioral and food consumption habits were notobserved in this study.

The compounds 1512, 3712 and 4112 of purity 95% or more (LC/MS) wereprepared for this study as 10% suspension in water containing 0.5% tween80.

B. Acute Oral Toxicity Test

The animals were distributed randomly into four groups (3 mice at eachgroup). One group did not receive any drug (control group). The secondgroup received an oral dose of 2000 mg/Kg of the drug and observed after24 h to count the deceased animals. According to the Guideline 423, whenall animals were found alive, the same test was repeated (dose 2000mg/kg) were given to the third group (3 animals) and observed for 24 h.All animals survived and therefore, a dose of 5000 mg/kg wereadministered to the fourth and final group.

The invention claimed is:
 1. A pharmaceutical composition havingcytotoxic activity against colorectal cancer cells, comprising: acompound of formula (I):

and a pharmaceutically acceptable carrier and/or excipient.
 2. Thepharmaceutical composition of claim 1, which is in the form of anaqueous suspension, and wherein the pharmaceutically acceptable carrierand/or excipient is a non-ionic surfactant in water.
 3. Thepharmaceutical composition of claim 1, which comprises 5 to 15 wt. % ofthe compound of formula (I), relative to a total weight of thepharmaceutical composition.